Cryopreservation of sturgeon sperm is a promising tool for aquaculture and conservation; however, its optimisation is limited by an incomplete understanding of how cryodamage affects spermatozoa that remain functionally competent after freezing and thawing. Most previous studies have focused on whole post-thaw samples, potentially masking cryopreservation-induced alterations that persist in motile spermatozoa. In this study, we investigated the effects of cryopreservation and post-thaw Percoll density gradient centrifugation (DGC), used to selectively isolate motile spermatozoa, on sperm kinematic parameters, membrane lipid composition, and fertilising ability in the sterlet (Acipenser ruthenus). Sperm motility was assessed using computer-assisted sperm analysis, sperm subpopulations were characterised by cluster analysis, and membrane lipid classes and fatty acids were assessed by thin-layer chromatography and gas chromatography, respectively. Cryopreservation reduced the proportion of motile spermatozoa, whereas DGC effectively separated a motile sperm fraction both before and after freezing. The kinematic properties of motile spermatozoa remained largely unchanged across treatments, indicating that Percoll DGC primarily removes non-motile or severely damaged cells rather than altering swimming behaviour. In contrast, cryopreservation induced pronounced alterations in lipid class and fatty acid composition, which persisted in the motile post-thaw sperm fraction. Despite this lipid remodelling, Percoll-selected cryopreserved sperm exhibited significantly improved fertilisation success, embryonic development, and larval quality. To our knowledge, this is the first study in fish demonstrating that cryopreservation-associated changes in lipid class and fatty acid composition persist in motile spermatozoa, and that post-thaw sperm fractionation can improve fertilising ability while reducing the risk of cryopreservation-associated developmental abnormalities in the progeny.

Abstract Background: Cryopreservation is a valuable tool in aquaculture and conservation programs, yet it exposes spermatozoa to physiological and molecular stresses that may impair motility, fertilisation capacity, and genomic stability. In fishes, where sperm motility is brief and easily activated, post-thaw separation of high-quality sperm remains technically challenging and poorly understood. This study evaluated whether density-gradient centrifugation can isolate a functionally superior subpopulation of sterlet (Acipenser ruthenus) spermatozoa with enhanced motility, fertilising ability, and reduced cryopreservation-induced epigenetic alterations. We further examined whether the use of this selected fraction influences DNA methylation patterns in resulting embryos. Results: Cryopreservation substantially reduced the proportion of motile spermatozoa, while density-gradient centrifugation consistently enriched motile cells both before and after freezing. Motility enhancement reflected a higher proportion of cells capable of activation rather than changes in kinematic behaviour. Fertilisation trials confirmed that the selected post-thaw fraction produced fewer malformed embryos compared with unselected cryopreserved sperm. Cryopreservation and post-thaw selection induced small but significant methylation changes in sperm, predominantly in intergenic regions and promoter-proximal elements. However, these epigenetic differences were not maintained in embryos. Embryo methylomes showed minimal variation between treatments, no distinct clustering by sperm origin, and negligible numbers of differentially methylated regions. Thus, although cryopreservation and sperm selection influenced sperm DNA methylation, these alterations did not translate into measurable changes in embryo methylation patterns. Conclusions: Density-gradient centrifugation effectively isolates a motile, functionally improved sterlet sperm fraction after cryopreservation, enhancing fertilisation outcomes and reducing developmental abnormalities. Cryopreservation and sperm selection introduce detectable but limited methylation variation in spermatozoa; however, these changes are not inherited by embryos. The findings highlight the utility of post-thaw sperm selection in aquaculture practice and indicate that cryopreservation-associated epigenetic variation in sterlet sperm does not propagate to early developmental stages. ### Competing Interest Statement The authors have declared no competing interest. Czech Science Foundation, https://ror.org/01pv73b02, GACR 22-14069S Ministry of Education Youth and Sports, https://ror.org/037n8p820, 90238

This study investigated the spermatogenesis of the freshwater stingray Potamotrygon motoro using microscopic analyses. The testes of this species were described as being composed of germinal zones, a degenerated zone, and the epigonal organ, consisting of connective tissue and lymphomyeloid cells. A cystic pattern of spermatogenesis was observed, in which each cyst is formed and maintained by Sertoli cells that undergo morphological and positional changes throughout the process. After the release of spermatozoa into the duct, the peripheral cysts formed a degenerating layer. Spermiogenesis, the final phase of sperm development and differentiation, was identified as a complex process in P. motoro, involving nuclear compaction and structural modifications. This study presents, for the first time, a detailed description of the germ cell development process in P. motoro, contributing to the understanding of spermatogenesis in freshwater stingrays.

Intracytoplasmic sperm injection (ICSI) is an emerging technique in aquaculture breeding and conservation programs, but its success depends on species-specific characteristics of egg activation and mechanical tolerance. In sturgeon, as in many teleosts, water exposure activates eggs independently of sperm, triggering chorion hardening, perivitelline space formation, and meiotic progression, changes that may affect the feasibility of microinjection. Defining the activation window during which oocytes remain suitable for ICSI is therefore essential for protocol optimization. We evaluated ICSI performance in sterlet (Acipenser ruthenus) eggs subjected to different activation durations. Eggs activated for 5-15 min remained competent to support development, whereas activation for >= 20 min caused increased internal pressure and chorion rigidity, greatly reducing successful microneedle penetration and increasing oocyte rupture. Non-activated eggs yielded the highest developmental rates, likely due to lower mechanical resistance and favorable nuclear-cytoplasmic conditions. Egg quality, influenced by maternal stress, also contributed to developmental variability. Nonetheless, ICSI with single fresh or cryopreserved sperm produced normal embryogenesis and hatching in briefly activated eggs, indicating that short pre-activation does not preclude successful development. To explore conservation applications, ICSI was performed using Percoll-separated, frozen-thawed beluga sturgeon (Huso huso) sperm stored for nine years. Both activated and non-activated sterlet eggs supported early embryo formation under these conditions. These findings define a workable activation window for sturgeon ICSI and provide a practical foundation for assisted reproduction techniques relevant to aquaculture production and the conservation of endangered sturgeon species.

Sperm subpopulations represent a fundamental manifestation of intra-ejaculate heterogeneity in fish, arising from differences in structural features, motility patterns, and physiological states of sperm cells. This variability is increasingly recognised as biologically significant, influencing fertilisation success, sperm competition, and egg selection. Advances in computer-assisted sperm analysis, flow cytometry, and fluorescent staining have enabled the identification of distinct subgroups based on kinematic, morphological, and biochemical traits. However, techniques for their physical separation and functional assessment remain in early development for fish, limiting their routine application in aquaculture. Understanding sperm heterogeneity offers innovative perspectives for reproductive biology, providing insights into species with complex mating strategies and guiding the refinement of aquaculture practices, particularly in situations where sperm quality is reduced or highly variable. Applications include optimising artificial fertilisation, improving cryopreservation protocols, and enhancing genetic stability by isolating high-quality subpopulations, especially when optimal volumes of high-quality sperm are not available. Importantly, sperm subpopulation analysis also holds promise for conservation programs, where selecting and preserving viable gametes is critical for maintaining genetic diversity and supporting the recovery of endangered and threatened fish species. By reducing abnormal larval development and improving fertilisation efficiency, this approach strengthens hatchery success and contributes to long-term sustainability. Overall, framing sperm heterogeneity as both a biological phenomenon and a biotechnological resource highlights its importance and innovation. This review synthesises current knowledge, explores possible origins of sperm subpopulations, and outlines future directions for their application in aquaculture and conservation, with particular relevance in contexts where reproductive performance is compromised or variable.

Paternal RNAs are involved in the phenotype of progeny that may be transmitted across generations. In fishes, sperm RNAs have been identified, however their sources and biological functions remain largely unknown. In this review, we highlight that sperm RNAs are the remnants of spermatogenesis or are obtained from extracellular vesicles in the sperm duct during maturation. Therefore, multiple factors can influence the sperm RNAs profiles by modulating these processes. Sperm RNAs are transferred to embryos at fertilization, resulting in the reestablishment of paternally acquired traits in offspring. This suggests a role for sperm RNAs in mediating RNA-dependent paternal inheritance across generations. These underlying mechanisms involve the modulation of gene cascades in the embryos or the reshaping of chromatin to different conformations. This review addresses current knowledge gaps regarding the regulatory mechanisms underlying RNAs biogenesis and modification in fish sperm. It suggests that further investigations are needed to elucidate the functional consequences of RNAs profiles and their modifications that contribute to paternal effects on offspring phenotypes, providing aquaculture, fisheries, and conservation with invaluable information.

Sperm cryopreservation represents a valuable tool in aquaculture for resource management, breeding, and preservation. This study explores the potential of cryopreservation in common carp breeding by comparing the performance and morphometry of progeny arisen from cryopreserved (CSG) or fresh (FSG) sperm until two years old reared under semi-intensive fishpond management. Offspring were produced using sperm from 50 males per group, fertilizing a pooled egg batch from 15 females. Fertilization and hatching success, and important growth-related traits such as body weight (BW), Fulton's condition factor (K), fillet yield predictor (E8/E23), muscle fat, and morphometry were determined via phenotyping and digitization. The fertilization and hatching rates of inseminated eggs significantly differed between the treatments in favor of the FSG. However, the hatching rate of the fertilized eggs and larval malformation occurrence were comparable. The survival rates during the first and second winter were significantly (pspace

Subclass Elasmobranchii belongs to an old evolutionary class of Chondrichthyes that diverged 450 mya, presenting a wide diversity of reproductive strategies while preserving the ancient mode of internal fertilization. Despite such evolutionary success, many species in this group are at serious risk of extinction. Understanding the principles of sperm progressive motility and physiology of such an ancient group of vertebrates is crucial for advancing future assisted reproductive techniques to safeguard this species and for deepening our understanding of the evolution of reproduction. Elasmobranchii species possess big spermatozoa (compared to bony fishes) with an elongated helical head and tail similar to one currently existing (but later diverged) in birds, reptiles, and amphibians, which can be considered an evolutionary ancient. These structures may be associated with the necessity to penetrate viscous ovarian fluid or the jelly layer of eggs, suggesting environmental viscosity as the driving pressure shaping large-sized sperm heads into helical shapes through evolution. We observed spermatozoa motility with high-speed video microscopy to capture sperm and flagellar motion in three Elasmobranchii species: the freshwater ray Potamotrygon motoro, the marine skate Raja asterias and the shark Scyliorhinus canicula. We investigated the effect of viscosity on spermatozoa motility parameters and its ability to break free from spermatozeugmata, move progressively, and perform directional changes. After 20 min of observation, the spermatozeugmata conserved their structure in a low viscosity media of 1000 mOsm/kg osmolality. In comparison, no remaining structure of spermatozeugmata could be found in high-viscosity media with 2% methylcellulose (MC) in all three species due to progressive spermatozoa motion. We find that spermatozoa's unique helical head-to-flagellum architecture is specific to promote locomotion in high-viscosity fluid; they cannot move progressively in low viscosity. The highest velocity for shark sperm was observed at 0.75% MC and 1% MC for ray and skate sperm. Viscosity stabilizes the flagellar propagation, producing rotational forces and allowing the helical head to "screw" into the media. Our observations suggest that the surrounding viscosity is critical to allowing spermatozoa progressive motility and enabling spermatozoa to control direction via newly observed head buckling in high viscosity. As such, the viscosity may be a key element controlling and regulating sperm performance and navigation during fertilization in the Elasmobranchii species.

ABSTRACTStudies have demonstrated adverse effects of global warming on aquatic ecosystems. However, the effects of increased water temperature (IWT) on fish reproduction are still doubtful in species spawning at low or high temperatures. In this meta‐analysis, we elucidated the effects of global warming on spermatozoa functions, key determinants of male fertility. We recruited 245 data records from 20 studies spanning 20 cold‐ and warm‐water fishes to identify the effects of IWT on sperm quality. Data were re‐processed and re‐analyzed to determine the overall effects of IWT on sperm kinetics such as percentage of motile spermatozoa (MOT), duration of spermatozoa motility (DSM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), as well as on enzymatic activities for energy supply (EAES) and antioxidant enzyme activity (ANEA). The standardized mean difference was calculated for each study, with positive values indicating higher performance under IWT. Results showed that (a) the overall effect size for MOT was larger negative in cold‐water fishes (−1.22) than in warm‐water fishes (−0.95). (b) Each 1°C increase in the activation medium reduced MOT by 1.30% (cold‐water fishes) and 3.47% (warm‐water fishes). (c) IWT negatively affected DSM, decreasing it by 10 s (cold‐water species) and 5.64 s (warm‐water species) per degree of IWT. (d) Spermatozoa velocity (VCL and VSL) increased by IWT in warm‐water species. (e) In cold‐water species, IWT‐induced detrimental effects on EAES were associated with adverse impacts on sperm motility kinetics. In conclusion, IWT negatively affects sperm motility kinetics, suggesting an impact of global warming on fish reproduction.

Although short-term hypothermic storage of sperm is already widely used in artificial fish reproduction and is considered very useful for resolving multiple problems related to logistics and broodstock maintenance, this approach continues to be explored and developed. In the present study, targeted and global metabolome LC/MS analyses of common carp sperm during refrigerated storage in undiluted and diluted (in a buffer: NaCl 55 mmol/L, KCl 40 mmol/L, CaCl2 1 mmol/L, MgSO4 0.5 mmol/L, sucrose 87.5 mmol/L, Tris 10 mmol/L, pH 8, with or without 5% methanol) conditions were used to find potential metabolic markers of fish sperm quality and storage capability evaluated by sperm motility parameters. Sperm motility percentage started to decline from the 6th day of the storage, followed by a significant drop in ATP content in all studied groups, ADP content in undiluted sperm, and creatine phosphate content in sperm diluted by the methanol-containing buffer. Correlation analysis of whole metabolome and sperm motility data allowed us to suggest several potential markers belonging to different chemical classes (mainly nucleotides and their components). Considering motility preservation and changes in the content of proposed markers of sperm quality/storage ability, sperm storage in the methanol-containing buffer is more promising for further application. At the same time, further searches for reliable markers and their validation remain necessary.

IntroductionThe successful cryopreservation of common carp sperm is crucial for its application in aquaculture and selective breeding programs. This study investigates the efficacy of cryopreserving sperm in large containers (5 mL) with a low dilution rate (1:1) in three different cryoprotective media and thawing in different conditions.MethodsThe developed method utilizes a low-ionic (hypotonic) cryoprotective medium, freezing with a controlled cooling rate, and high-temperature sperm thawing (60°C). The investigation employs a detailed spermatozoon motility assessment.ResultsPost-thaw motility of 32.3% ± 14% and initial curvilinear velocity of 89 ± 20 μm/s across 30 males were observed. Principal component analysis of sperm kinematic characteristics revealed distinct populations of sperm cells exhibiting varying responses to cryopreservation. The developed method achieved successful fertilization comparable to that of the non-frozen control group using sperm from a single cryotube (2.5 mL, approximately 50 * 109 spermatozoa) to fertilize 200 g of eggs (1:120,000 egg:spz).DiscussionThis novel approach demonstrates an effective cryopreservation protocol for common carp sperm in large-volume cryo-containers in combination with low-ionic cryomedia and high thawing temperature, providing methods well-suited for fisheries practices and selective breeding programs. Future studies of the biological properties of different sperm subpopulations in post-thaw sperm samples can contribute to a deeper understanding of sperm biology, improve cryopreservation techniques, and enhance the success rates of assisted reproductive technologies.

The primary function of spermatozoa is to fertilize the oocyte, which depends on their motility and is directly associated with their metabolic state. The oxygen consumption rate (OCR) of spermatozoa reflects the respiratory capacity of sperm mitochondria under various physiological conditions and is an essential marker of sperm quality. We determined the OCR of common carp (Cyprinus carpio) sperm using two respirometry methods: the conventionally used polarographic method with a Clark-type electrode and fluorometric assay with an Oxo Dish optochemical oxygen sensor. The latter was used for the first time to evaluate spermatozoa oxygen consumption in various metabolic states (under different treatments) at different dilution rates. These two methods were compared using Bland-Altman analysis, and the applicability of the optochemical oxygen sensor for evaluating carp sperm oxygen consumption was discussed. Sperm motility and progressive velocity parameters were also assessed to evaluate the effect of sperm respiration under different metabolic states and dilution rates and preincubation period on the physiological status of spermatozoa. The comparison of these respirometry methods clearly shows that while the polarographic method allows immediate measurement of oxygen levels after adding a sperm sample, the optochemical oxygen sensor has a priority in the amount of data obtained due to simultaneous measurements of several samples (e.g., different males, different fish species, repetitions of the same sample or various experimental conditions), even at a later time after adding sperm to the measuring chamber. However, the compared methods are complementary, and the proposed methodology can be applied to other fish species.

Spermatozoa motility kinetics have been found to increase in short-term stored sperm on ice of common carp (Cyprinus carpio) following incubation at a high temperature (20 degrees C) for 10-20 min. The aim of the present study was to elucidate the contribution of the ATP store and plasma membrane function in temperature -induced motility kinetics of short-term stored sperm. Spermatozoa ATP content significantly decreased after 9 days of short-term storage on ice; however, it remained unchanged after incubating stored sperm at higher temperatures. Spermatozoa motility kinetics of either fresh or stored sperm on ice for 14 days were enhanced following incubation or activation at 20 degrees C. The motility of demembranated spermatozoa from 14 days of stored sperm could be nearly fully activated, similar to that of fresh spermatozoa. The swelling ratio of spermatozoa incubated and activated at high temperatures was higher than that at low temperatures. Spermatozoa swelling was observed to be faster from sperm stored for 14 days than fresh sperm at the same incubation and activation temperature. These results suggest a physiological contribution of the plasma membrane to enhance spermatozoa motility kinetics in stored sperm following incubation at 20 degrees C.

Studies have demonstrated adverse effects of global warming on aquatic ecosystems from the abiotic to the biotic level. In the present work, a meta-analysis study was conducted to elucidate the effects of global warming on spermatozoa functions, which are key determinants of male fertility. We recruited 245 data records from a pool of empirical studies, which includes 20 studies spanning 20 cold- and warm-water fish species, to identify the effects of increased water temperature (IWT) on determinants of sperm fertility in fishes. Data were systematically re-processed and re-analyzed to determine the overall effects of IWT on sperm kinetics such as motility (MOT), duration of motility (DSM), curvilinear velocity (VCL), rectilinear velocity (VSL) and average path velocity (VAP), as well as on enzymatic activities for energy supply (EAES) and antioxidant enzyme activity (ANEA). The standardized mean difference was calculated for each study, with positive values indicating higher performance under IWT. Results showed that (a) the overall effect size for MOT was more negative in cold-water fishes (-1.22) than in warm-water fishes (-0.98). (b) Each 1 °C increase in the activation medium reduced MOT by 1.30% (cold-water fishes) and 3.47% (warm-water fishes). (c) The IWT negatively affected DSM, decreasing it by 10 s (cold-water species) and 5.64 s (warm-water species) per degree of IWT. (d) Spermatozoa velocity (VCL and VSL) was increased by IWT in warm-water species. In conclusion, this study shows that IWT negatively affects sperm motility kinetics, suggesting an impact of global warming on fish reproduction.### Competing Interest StatementThe authors have declared no competing interest.

To avoid mechanical compression of spermatozoa during the cryopreservation, a high dilution ratio with the cryoprotective medium is applied. Due to this, the sample volume increases, which entails increased cryobank capacity. Moreover, the high volume of cryomedias does not allow to fertilize of large volumes of eggs in practical artificial reproduction of common carp aquaculture. The current study demonstrated that reasonable lowering of dilution rates might still be effective for carp sperm cryobanking & mdash;elevation of spermatozoa con-centration up to 13 x 109 spz mL-1 resulted in a significant decrease in post-thaw sperm motility percentage to 20% compared with 39% in 0.5 x 109 spz mL-1. Nevertheless, no significant differences in sperm kinetic pa-rameters (VCL and LIN) were found in this case. The fertilization outcome (embryo development and hatching rates) was similar after applying thawed sperm samples with & ldquo;optimal & rdquo; (2.2 & ndash;2.4 x 109 spz mL-1) and & ldquo;sub-optimal & rdquo; (11.0 & ndash;13.0 x 109 spz mL-1) concentrations (sperm/egg ratio at fertilization was in the range of 0.3 & ndash;4.5 x 105 spz/egg). Thus, applying a low dilution rate such as one part of sperm to one of the cryoprotective me-diums is favorable for decreasing cryo-storage space and the sperm volume needed to fertilize big egg numbers. The experiment also shows that the 4.5 x 105 spz/egg ratio is not sufficient for good fertilization, and it is necessary to use higher sperm concentrations per egg or improve the method of fertilization. Superscript/Subscript Available

This study studied the possibility of sperm reactivation employing second motility activation, adenosine triphosphate (ATP) reloading, and regaining the fertilizing ability and the possibility of improvement in the fertilisation rate of reactivated sperm by supplementation with metabolites. The most crucial bioenergetic pathways during quiescent, motile, and reactivated states of sterlet (Acipenser ruthenus) spermatozoa were determined. Results showed that out of the studied pathways (glycolysis, mitochondrial respiration, and phosphocreatine-creatine – PCr-Cr shuttle system), PCr-Cr shuttle system was the most requested during the quiescent state. While after motility activation, no studied pathway had a significant energy supply contribution. Thus, the bioenergetic strategy to sustain sperm motility in sturgeon is similar to that in teleostean fishes. After termination of the first round of motility, the spermatozoa of sterlet were able to become motile and fertilise the egg cells after 1-h incubation in a reactivation medium. Mitochondrial respiration and the PCr-Cr shuttle system were shown to be active during the reactivation state. However, unlike spermatozoa of teleostean fishes, the sterlet reactivated spermatozoa did not demonstrate ATP reloading. This may indicate for the first time that the bioenergetic strategy during second motility activation in sturgeon sperm is divergent from cyprinids and salmonids. The fact that only part of spermatozoa can be reactivated is probably related to the existence of sperm subpopulations of specific intrinsic physiological properties. Further study of these properties will enhance our fundamental knowledge in fish spermatology and help increase the effectiveness of artificial fish reproduction.

Fish speciation was accompanied by changes in the urogenital system anatomy. In evolutionarily modern Teleostei, male reproductive tracts are fully separated from the excretory system, while in evolutionarily ancient Chondrostei and Holostei, the excretory and reproductive tracts are not separated. Sturgeon post-testicular sperm maturation (PTSM) occurring as a result of sperm/urine mixing is phenomenologically well described, while, in holosteans, functional intimacy of seminal ducts with kidney ducts and the existence of PTSM still need to be addressed. In Lepisosteus platostomus (Holostei), sperm samples were collected from testes (TS), efferent ducts (EDS), and Wolffian ducts (WDS). While WDS was motile, no motility was found in TS and EDS. The existence of PTSM was checked by in vitro PTSM procedure. After TS and EDS incubation in seminal fluid from WDS, no more than 5% motile spermatozoa were observed in TS, whereas in EDS the motility percentage was up to 75%. Experimental dyeing of urogenital ducts in gars and sturgeons revealed some differences in the interconnection between sperm ducts and kidneys. It is concluded that post-testicular sperm maturation occurs in gars and suggests that infraclass Holostei occupies an intermediate evolutionary position between Teleostei and Chondrostei in the anatomical arrangement of the urogenital system.