Anatolii Sotnikov

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.

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.

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.

Introduction: The 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.Methods: The 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.Results: Post-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).Discussion: This 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.

Hormone treatments are routinely employed with bala shark Balantiocheilos melanopterus male broodstock to stimulate the production of high-quality sperm. In the current trial, three spermiation-inducing treatments were evaluated: 20 μg/kg body weight (BW) recombinant hCG; 20 μg/kg BW [D-Ala⁶, Pro⁹, NEt]- gonadotropin-releasing hormone (GnRHa) with 20 mg/kg BW metoclopramide; and poly(lactic-co-glycolic acid) (PLGA) microparticles with slow release of 10 μg/kg BW GnRHa. A 0.9% saline AS negative control was also included. Administration of the GnRHa through the form of slow release of PLGA microparticles 24 h after treatment resulted in a significantly higher sperm volume, motility percentage, and total sperm count compared to the control. Injection of GnRHa with metoclopramide induced sperm parameters that did not differ from the control, with the exception of motility percentage. The lowest potency to induce spermiation in bala sharks was in the treatment with recombinant hCG. Both PLGA microparticles and GnRHa with metoclopramide significantly increased blood plasma concentrations of testosterone and 11-ketotestosterone compared to the control. The PLGA microparticle system with continuous release of 10 μg/kg BW GnRHa was the most effective treatment in inducing spermiation in bala sharks.

Carp pituitary treatment versus poly (lactiac-co-glycolic acid) microparticles with slow release of Alarelin at 35 µg kg⁻¹ or 200 µg kg⁻¹ body weight to induce spermiation was compared in sterlet Acipenser ruthenus. All hormone treatments initially increased testosterone and 11-ketotestosterone, with a subsequent decline in testosterone but consistent high levels of 11-ketotestosterone at 48 and 72 h post-treatment. Spermiation did not differ between hormone-treated groups, and was not detected in controls receiving saline solution. Administration of the carp pituitary led to maximum sperm production 24 h post-treatment, followed by a decrease at 48 h post-treatment, with no sperm obtained at 72 h. The effect of Alarelin at 35 µg kg⁻¹ bw and carp pituitary did not differ at 24 and 48 h post-treatment, whereas 200 µg kg⁻¹ bw Alarelin was associated with significantly lower spermatozoon concentration 24 h post-treatment compared to carp pituitary, with no difference in milt volume. Higher relative sperm production was observed 48 h after injection of Alarelin at 200 µg kg⁻¹ bw compared to carp pituitary. Spermatozoon motility was significantly higher in fish receiving Alarelin at 35 µg kg⁻¹ bw than 200 µg kg⁻¹ bw. The treatment with optimal effect on inducing spermiation was poly (lactic-co-glycolic acid) microparticles with slow release of Alarelin at 35 µg kg⁻¹ bw.