Carp Sperm Research Articles

Insecticides Cypermethrin and Chlorpyrifos Exposure: Effects on Sperm Motility and Fertility in Cyprinus carpio (Cypriniformes, Cyprinidae)

The agricultural production of the Argentine Pampa is based on intensive cultures, and the most widely used insecticides correspond to cypermethrin (pyrethroid) and chlorpyrifos (organophosphate), among others. Sperm motility and fertility assessments are essential topics for biology and population dynamics studies on fish species. In this study the alterations of these endpoints after exposure to commercial formulation of both insecticides were used to analyze the effects on common carp sperm. After the in vitro exposure at nominal concentrations of 0.25, 0.5, 1, and 2 μg/L of cypermethrin, a significant reduction in motility in 0.5 μg/L concentrations was observed respect control group. The same nominal concentrations were tested for chlorpyrifos, and the same significative effect was detected at 2.0 μg/L. Fertilization rate was significantly reduced at concentrations from 0.5 μg/L cypermethrin exposure, and from 1.0 μg/L in the case of chlorpyrifos. Eggs LC50 / 24 h (95% confidence limit) exposed to cypermethrin was 0.57 μg/L (0.36 - 0.88), and exposed to chlorpyrifos was 2.45 μg/L (1.13-5.29). However, is still debatable how these insecticides impact on fish reproduction, using this in vitro scenario we found a clear reduction in sperm motility and fertility.

Common carp sperm chromatin as an economical and effective remover for benzo(a)pyrene from pollutants

Benzo (a) pyrene is a highly carcinogenic polycyclic aromatic compound, difficult to be degraded, widely present in the environment. However, there is currently no cost-effective and efficient method for removing benzo (a) pyrene. In this study, a feasible method was introduced to cheaply and efficiently adsorb benzo(a)pyrene using chromatin. Scanning electron microscopy analysis showed that the chromatin had a filamentary fiber structure. Fourier transform infrared (FTIR) spectroscopy showed that benzo(a)pyrene formed a bond with the chromatin. Effective binding was confirmed using fluorescence microscopy. Influence factors exploration experiments indicated that the amount of benzo(a)pyrene adsorbed by chromatin was 0.16 mg g−1. The adsorption process of BaP by chromatin is consistent with a pseudo-second-order kinetics model of adsorption. The adsorption isotherm model is consistent with the langmuir isotherm model.This study suggests that chromatin can be utilized as a ordinary and high efficiency adsorbent for removing benzo(a)pyrene and can be utilized in further studies.

A new method for creating androgenetic diploid fish from tetraploid carp sperm and their genetic composition analysis

The application of cold shock to inhibit the extrusion of the second polar body in fertilized eggs is one of the main methods to induce triploid in diploid fish. In this study, we originally planned to induce hexaploid by this method in tetraploid carp. However, among the surviving offspring, we observed only 50.7% tetraploid and 49.3% diploid individuals, which could potentially result from either androgenetic diploids or gynogenetic diploids. The absence of irradiation and the high proportion of diploids attracted us. However, the lack of sex-specific markers for tetraploid carp, makes it difficult to determine their origin. Fortunately, we also carried out the cross of yellow carp (Cyprinus carpio yellow var., 2n, ♀) × tetraploid carp (♂) and red crucian carp (Carassius auratus red var., 2n, ♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis, diploid offspring have also been found, and whether they have undergone gynogenesis or androgenesis can be judged based on phenotype and genotype. In terms of phenotypes, the key phenotypes of two diploids, such as the whisker length, number of lateral scales, and presence of melanin, are consistent with tetraploid carp. In terms of genotypes, through single-copy nuclear gene cloning and fluorescence in situ hybridization (FISH), it is supported that both diploids are androgenetic offspring. In addition, 14.9% of tetraploid offspring were found in red crucian carp (♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis. By the above-mentioned means of detection, it was found that they were created by inhibiting the extrusion of the second polar body of the fertilized eggs. From this it will be seen that the application of cold shock before the extrusion of the second polar body of fertilized eggs cause not only polyploidy, but also the elimination of both second polar body and female nucleus. Previous studies have found that cold shock can induce lethal androgenetic haploids in diploid fish, but these androgenetic diploid fish produced by tetraploid carp sperm were surviving. We summarize the mechanism behind the above phenomenon, which is mainly related to meiosis occurring at the edge of the cell membrane. Cold shock may disrupt the function of the spindle, resulting in the simultaneous retention or extrusion of female nucleus and second polar body. This study will provide valuable insights into androgenesis, polyploidization, and hybridization in fish genetic breeding.

Comparative transcriptomic analysis of the brain-liver Axis reveals molecular mechanisms underlying acute cold stress response in Gynogenetic Mrigal carp

In this study, we produced a population of gynogenetic mrigal carp (Cirrhinus mrigala, GMCJ) through artificial gynogenesis using UV-inactivated white crucian carp spermatozoa. The GMCJ exhibited a faster growth rate and strong resistance to low temperatures, surviving natural winter conditions with temperatures below 10 °C. To gain a deeper insight into GMCJ's molecular response to acute cold stress (a decrease in water temperature from 26 °C to 14 °C within 1 h at a rate of 0.2 °C/min), we conducted a comparative analysis of the histology and transcriptomics of the brain-liver axis. Transmission electron microscopy of the heart tissues revealed that acute cold stress result in widened myofilament spacing, along with enlarged, deformed mitochondria that developed vacuoles and ruptured. We identified differentially expressed genes (DEGs) in the brain (2463 upregulated and 3288 downregulated genes) and liver (1210 upregulated and 1224 downregulated genes). Analysis of the DEGs' correlations indicated that major pathways involved in immunity, disease, metabolism, and growth were active and interconnected. KEGG functional analysis showed that the most enriched pathways in the brain-liver axis involved the regulation of the glucagon pathway, PI3K-Akt signaling pathway, and herpes simplex infection. Numerous genes associated with the nervous-endocrine system were activated, suggesting a coordinated response of the brain-liver axis to acute stress. These findings enhance our understanding of how farmed, particularly gynogenetic fish respond to acute cold stress.

The Effect of Storage Time on The Quality of Common Carp Sperm (Cyprinus carpio) Strain Punten in NaCL Solution and Coconut Water (Cocos nucifera L.) Extender

The Effect of Storage Time on The Quality of Common Carp Sperm (Cyprinus carpio) Strain Punten in NaCL Solution and Coconut Water (Cocos nucifera L.) Extender

Do milt storage techniques affect the quality of carp spermatozoa during short-term storage?

Short-term storage and management of milt are crucial parts of hatchery practice. There is speculation that sperm stored in a small Eppendorf could not retain their optimal performance compared to those stored in a 50 ml cell culture flask (CCF), thus raising the question of discrepancy between laboratory and hatchery conditions. Therefore, this study aimed to compare sperm quality parameters, such as osmolality and the ionic composition of seminal plasma, spermatozoa motility and its ability to maintain the volume under various osmotic conditions (310, 230, 150, 90, and 20 mOsm/kg) in fresh sperm and after its storage for 24 h in 1.5 ml Eppendorf or 50 ml CCF at 4 °C without any extender. Freshly collected sperm had higher motility percentages at each osmotic condition than 24 h stored sperm in Eppendorf and CCF. There was no significant difference in spermatozoa motility at various osmotic conditions except 90 mOsm/kg osmolality. Besides, VCL, VSL, VAP, and LIN of spermatozoa, osmolality, and ionic composition of seminal plasma did not significantly differ between these two containers. This study suggested no difference between sperm stored in an Eppendorf or CCF regarding sperm functionality.

Cryopreservation of sperm from the gudgeon, Microphysogobio rapidus (Cyprinidae): Effects of cryoprotectant, diluents, and dilution ratio

We investigated methods for cryopreserving sperm from the endangered gudgeon, Microphysogobio rapidus, by examining the effects of cryoprotective agent (CPA) concentration, diluent, and dilution ratio on post-thaw sperm quality. The quality of frozen sperm was evaluated in terms of motility and kinematic parameters, viability, DNA damage, and fertilization rate. We evaluated methanol, glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol as CPAs. Sperm motility, velocity, and viability were significantly higher when methanol was used as the CPA (p < 0.05). The diluents tested were Ringer's solution, Kurokura's Extender, Common Carp Sperm Extender (CCSE), and buffered sperm motility-inhibiting saline solution (BSMIS); post-thaw motility was highest when Ringer's solution was used as the diluent. Next, various quantities of methanol were combined with Ringer's solution to identify the optimal dose of methanol. The dilution ratios tested ranged from 1:1 to 1:7. Cryopreserved sperm was thawed at 20 °C for 15 s. The use of 10% methanol with Ringer's solution at a dilution ratio of 1:5 resulted in the highest post-thaw sperm motility, viability, and velocity including VAP, VCL, and VSL. Post-thaw sperm showed significantly greater DNA damage than the control (fresh sperm) (p < 0.05). The fertilization rate was highest with fresh sperm (p < 0.05), followed by sperm frozen with 10% methanol + Ringer's solution. We recommend that the best way to preserve sperm in the studied species is to use a combination of Ringer's solution and 10% methanol at a 1:5 dilution ratio. Our findings will facilitate the artificial fertilization of M. rapidus.

Optimizing aquaculture-scale common carp artificial reproduction: a novel approach to sperm cryopreservation using large-volume containers and elevated thawing temperatures

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.

Changes in sperm metabolome during carp sperm short-term storage in different media: In search of sperm quality and storage capability markers

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.

The growth performance of pond-reared common carp (Cyprinus carpio) larvae propagated using cryopreserved sperm.

The aim of our study was to determine the efficacy of utilizing cryopreserved common carp sperm (in comparison to fresh sperm) for propagation at a Hungarian aquaculture facility. The sperm was frozen in 5mL straws using an extender method that was previously tested in common carp. Sperm motility was monitored using a computer-assisted sperm analysis system. The hatching and malformation rates among the specimens were recorded before the stocking of larvae in both groups. The growth (body weight, total length) and survival rates of the fish were measured during the pre-nursing (from May to June: between 1 and 26days post hatching) and grow-out periods (from June to October: between 26 and 105days post hatching) of the same year. The fresh sperm, which was collected and pooled prior to fertilization, showed high MOT (97%), pMOT (92%), VCL (106µms-1), LIN (75%), and ALH (1.84µm). Prior to the fertilization trial of the cryopreserved sperm, low MOT (34%), pMOT (14%), and VCL (61µms-1) values were observed in frozen-thawed sperm. A significantly higher hatching rate was measured in the fresh sperm group (87%) when compared to the cryopreserved sperm group (42%). No significant difference in the overall malformation rate was observed in larvae originating from either the fresh or frozen sperm. A significant difference between the two test groups was observed in the incidence of deformed tails (fresh: 20%, cryopreserved: 55%). Except for one sampling period, no significant difference in the body weight and total length of the fish larvae was found between the two groups throughout the pre-nursing and grow-out periods. A significantly higher larvae survival rate was noted in the fresh sperm (72%) as compared to the cryopreserved group (43%) by the end of the pre-nursing stage. However, no significant difference in survival rate was observed for the cryopreserved sperm (96%) in comparison to the fresh sperm (95%) by the end of the grow-out stage. The results of this study showed, for the first time in large-scale pond culturing, an equal growth and viability in larvae propagated from cryopreserved sperm when compared to fresh sperm (despite the limited available rearing ponds provided by the commercial company).

Optimisation of cryovials and milt dilution ratios to upscale carp sperm cryopreservation for use in hatchery seed production

This study reports the experimental results carried out to optimise containers and miltdilution ratios for carp sperm cryopreservation, in order to make the process simple andcompatible with the needs of commercial carp seed production, which is a small-scale andlow technology sector in Asia. The procedure optimisation for up-scaling was assessedby the ability of cryopreserved sperms to fertilise Cyprinsu carpio eggs and hatchingpercentage, in three different experiments. In the first experiment, we tested the effect ofmilt to different extender dilutions (1:4, 1:6 and 1:8); the second experiment tested theeffect of container capacities (0.5 ml French straw; 2 ml and 5 ml cryovials) with two dilutionratios (1:3 and 1:6) and in the third experiment, the efficacy of pre-mix of extender salts andindividually pre-weighed salts stored over 5 months was compared with fresh preparationof extender. Out of the three dilutions tested, two dilutions viz., 1:4 and 1:6, yielded hatchingabove 50%. We also used the milt cryopreserved at dilution of 1:3 which was post-thawdiluted with extender, to make final dilution of 1:6, to fertilise the eggs. This post-thawdilution in the ratio 1:6, yielded the results comparable to the milt cryopreserved in 1:6dilution. This approach can help small-scale seed producers to store more sperm withinthe limits of the cost of liquid nitrogen. The extender salt composition, pre-weighed andpre-mixed, was also found to give comparable results to the fresh composition after storageover 5 months. This can assist in developing ready to use, cost-effective, working kitsavailable to semi-technical hatchery seed producers. The parameters optimised in the studyhas potential to be transformed as an easy strategy with the use of 5 ml vial, cryopreservedat 1:3 dilution, with further post-thaw dilution at site, thus aiding in fertilisation of largevolume of eggs produced by high fecund carp species within the limited time available formaintaining good gamete quality. Keywords:Cryobanking, Cryovials, Dilution, Salt premix

Short‐term storage of sperm in common carp from laboratory research to commercial production—A review

AbstractShort‐term storage of fish sperm is a simple and cost‐effective process in fishery research from basic to applied perspectives. It has been used to elucidate and evaluate gamete biology and quality, perform in vitro fertilization, develop sperm cryopreservation. Sperm short‐term storage is used in selective breeding, hatchery production and advanced molecular investigations such as disease detection of commercial species in aquaculture as well as biological conservation of threatened or endangered fishes. Several studies have demonstrated that the quality of common carp Cyprinus carpio sperm can be influenced by their health, age, nutritional status, the dose of administered hormone and environmental factors. When sperm is stored for short time, spermatozoa motility and viability as key determinants of fertility affected by storage parameters including the composition of natural seminal plasma, duration of exposure, temperature, dilution ratio if the extender is used, and additives in addition to the factors that affect sperm quality such as the age of male and spawning period. This is due to damage to the integrity of the plasma membrane, production of reactive oxygen species (ROS) causing oxidative stress and epigenetic modification. This review highlights and investigates the recent data regarding worldwide aquaculture production, short‐term storage of common carp sperm and the effects of storage on spermatozoa function with consideration to factors that affect sperm quality during storage. It also highlights the knowledge gap that must be filled in the future for designing methods from laboratory into practical applications of short‐term storage in the breeding program of common carp.

Aging of common carp (Cyprinus carpio L.) sperm induced by short-term storage does not alter global DNA methylation and specific histone modifications in offspring

Short-term storage of sperm is a useful tool to synchronize the availability of male and female gametes for fertilization in a hatchery. Induced aging by short-term storage has been shown to affect fish spermatozoa phenotypes including morphology and motility kinetics. However, its effects on epigenetics of sperm and the resulting embryos have not been investigated. In the present study, sperm of common carp (Cyprinus carpio) was stored in vivo and/or in vitro using an extender, and spermatozoa motility kinetics, viability, and epigenetics were analyzed. We observed that spermatozoa motility and velocity decreased following in vivo and/or in vitro storage from 3 to 6.5 days, but spermatozoa viability remained stable. At the same time, global DNA methylation, DNA hydroxymethylation, and specific histone acetylation and methylation were not affected by sperm aging. Further analysis showed no variation in global DNA methylation during embryogenesis when short-term stored sperm in the extender was used for fertilization compared with embryos produced from fresh sperm. The results suggest the reliability of short-term storage of sperm using an extender in fertilization and the production of the next generation of embryos. However, larval growth and performance need to be elucidated for these fish. This study has shed light on the potential application of extenders in storage of sperm in common carp breeding programs to manage selection, genomic manipulation and genetic resources conservation.

Oxidative stress and DNA fragmentation in frozen/thawed common Carp Cyprinus carpio sperm with and without supplemental proteins

Using cryopreservation techniques can increase the effectiveness of reproducing cultured fish species by ensuring a dependable supply of sperm, although the quality of the sperm could be impacted by the procedures involved. The goal of this study was to investigate the effect of purified seminal plasma transferrin (Tf), bovine serum albumin (BSA), and antifreeze protein (AFP) types I and III at 1 µg mL−1 on relevant characteristics of cryopreserved sperm from common carp Cyprinus carpio. We compared oxidative stress indices, antioxidant activity, and DNA fragmentation of fresh sperm to that frozen with extender only or with Tf, BSA, or AFP types I and III. Fresh sperm had significantly lower levels of thiobarbituric acid reactive substances (TBARS) compared to samples that underwent cryopreservation without protein treatment, which resulted in 0.54 ± 0.06 nmol/108 cells of TBARS. Carbonyl derivatives of proteins (CP) decreased significantly (ANOVA; P > 0.05) in carp sperm with addition of Tf, AFPI, and AFPIII. Significant differences in superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) activity were seen in sperm supplemented with Tf, BSA, AFPI, and AFPIII from those without. Significantly less DNA damage, expressed as percent tail DNA (11.56 ± 1.34) and olive tail moment (0.59 ± 0.13), was recorded in samples cryopreserved with Tf. The findings indicated that addition of Tf, BSA, AFPI, or AFPIII to cryopreservation medium is beneficial to sperm preservation. The mechanisms through which these proteins act positively on sperm need to be further investigated.

The formation and study of allogynogenesis Hemibarbus maculatus Bleeker

Hemibarbus maculatus Bleeker (HM) is an economically important fish that is highly valued by consumers. Gynogenesis is an important breeding method in aquaculture that has been widely applied to many fish species. In this study, we adopted an improved method of gynogenesis that proceeds as follows: first, inactivated koi carp sperm were used to stimulate eggs of HM, and then the UV-inactivated koi carp (KOC) sperm and HM eggs were mixed and stirred for 20–30 s to activate the eggs. The activated eggs were placed in yellow mud at 0 °C for 20–22 min for cold shock treatment, and then placed in water at 4–6 °C, 8–10 °C, and 15–18 °C in order, each for 1–2 min. After incubation at 22–25 °C, gynogenetic Hemibarbus maculatus Bleeker (GHM) were obtained. Morphological measurements and blood cell comparisons showed that there were no significant differences between GHM and HM. At the molecular level, comparative analysis of microsatellites DNA indicated that one fragment of GHM came from the KOC. This study provides the first evidence that DNA fragments from male parents exist in GHM at the molecular level. This discovery has important implications for fish genetic breeding.

Elevated temperature promotes spermatozoa motility kinetics and fertilizing ability following short-term storage: An implication for artificial reproduction of common carp Cyprinus carpio in a hatchery

Short-term storage of sperm in an extender at a low temperature has been widely applied in hatchery management. In the present study, for the first time, we examined the effects of elevating temperature on spermatozoa motility kinetics and fertilization ability after short-term storage at 0–2 °C. Sperm of common carp (Cyprinus carpio L.) was diluted with an extender (110 mM NaCl, 40 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, pH 7.5, 310 mOsmol/kg) at the ratio 1:1 (v/v) in containers and stored for 6–9 days at 0–2 °C under aerobic conditions. At the end of storage, the sperm suspension was incubated at 20 °C for >10 min and spermatozoa motility, velocity [straight-line velocity (VSL) and curvilinear velocity (VCL)], viability, and fertilizing ability were recorded. Spermatozoa motility of sperm stored for 6 days was significantly increased after incubation at 20 °C for 10 and 20 min and activation in both distilled water and a saline solution (45 mM NaCl, 5 mM KCl, 30 mM Tris, pH 8.0). Elevating the temperature did not affect spermatozoa viability. Spermatozoa motility, VSL and VCL, and viability were decreased at 3 days post storage (DPS) after activation in distilled water without incubation at 20 °C. However, elevating the temperature for 10–20 min at any time post sperm storage (3, 6 or 9 DPS) could promote spermatozoa motility kinetics. For sperm stored for 9 days, fertilization and hatching rates were increased when it was incubated at 20 °C for 10 and 20 min prior to fertilization at the ratio 30,000 spermatozoa per egg. Increasing the spermatozoa to egg ratio to 100,000:1, however, masked the temperature effects on fertilization and hatching rates. Finally, a large-scale experiment was performed in hatchery in which sperm stored for 8 days was incubated for 2 h at 20 °C, resulting also in an increase in spermatozoa motility and fertilizing ability. Results of the present study show that elevating the temperature following short-term storage of common carp sperm at low temperatures improved spermatozoa motility kinetics and their fertilizing ability. This method may contribute to developing artificial reproduction of economically important fish species in aquaculture.

Protocol for common carp sperm cryopreservation for aquaculture-scale artificial reproduction

Protocol for common carp sperm cryopreservation for aquaculture-scale artificial reproduction

Protective Effects of Different Egg Yolk Sources on Cryopreservation of Scaly Carp (Cyprinus carpio) Sperm

Egg yolk is one of the most widely used cryoprotective components of extenders, especially for the cryopreservation of mammalian species’ sperm cells. However, there is a lack of information regarding their efficacy in cryopreservation of fish sperm cells. Thus, the objective of this experiment was to compare the effectiveness of egg yolk from different avian species (duck, goose, and chicken) on post-thaw quality and fertilization ability of scaly carp (Cyprinus carpio) semen following cryopreservation. Sperm samples diluted with the sucrose-based extender at the ratio of 1:10 were supplemented with 10, 15, and 20% ratios of different avian egg yolks. In the control group, sperm samples were diluted with the sucrose-based extender, without egg yolk. Following dilution, sperm samples were equilibrated at +4⁰C for 10 min and aspirated into 0.25-ml straws. Then, sperm samples were frozen 3 cm above the liquid nitrogen (LN2) surface and plunged directly into the LN2. The frozen sperm cells were thawed in a water bath at 35 ⁰C for 30 s and fertilization was carried out using a 1x105 spermatozoa/egg ratio. Based on the results, supplementation of sperm cells with 20 % duck egg yolk in a sucrose-based extender exhibited the best post-thaw progressive motility (67.8 ±1.24%), progressive motility duration (32.6 ±1.45 s), viability (82.4 ±1.36%), and fertility (92.6 ±1.28%) compared to the control group (P&amp;lt;0.05). The results of the experiment showed that duck egg yolk could be used as an alternative instead of chicken egg yolk in a sucrose-based extender for the cryopreservation of scaly carp sperm.

High sperm concentration during cryopreservation decreases post-thaw motility percentage without compromising in vitro fertilization outcomes in common carp

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 —elevation of spermatozoa concentration up to 13 × 109 spz mL−1 resulted in a significant decrease in post-thaw sperm motility percentage to 20% compared with 39% in 0.5 × 109 spz mL−1. Nevertheless, no significant differences in sperm kinetic parameters (VCL and LIN) were found in this case. The fertilization outcome (embryo development and hatching rates) was similar after applying thawed sperm samples with “optimal” (2.2–2.4 × 109 spz mL−1) and “sub-optimal” (11.0–13.0 × 109 spz mL−1) concentrations (sperm/egg ratio at fertilization was in the range of 0.3–4.5 × 105 spz/egg). Thus, applying a low dilution rate such as one part of sperm to one of the cryoprotective mediums 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 × 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.

Induction of gynogenesis with ultra-violet irradiated Koi carp (Cyprinus carpio haematopterus) sperm demonstrates the XX/XY sex determination system in Opsariichthys bidens

Opsariichthys bidens (Chinese hooksnout carp) is a small stream fish with high economic value. As the males of this species grow faster than females, it follows that mono-sex breeding has significant potential for commercial production. However, little is known about the sex determination system in O. bidens, this is the key process that is used to develop mono-sex breeding in fish. In the present study, we induced gynogenesis by fertilizing O.bidens eggs with ultra-violet (UV)- inactivated Cyprinus carpio haematopterus (Koi carp) sperm followed by cold shock treatment. Results showed that the optimal induction conditions was 25 min duration of cold shock treatment at 2 min post fertilization (mpf), the highest fertilization rate and hatching rate for gynogenic embryos were 68.24 ± 3.75% and 12.54 ± 1.59%, respectively. Embryos derived from gynogenesis developed normally, but the hybrid embryos failed (created with non-UV-inactivated Koi carp sperm and O.bidens eggs). Genetic analysis demonstrated only maternal inheritance in the gynogenetic offspring. Histological analysis further showed that the gonads of all gynogenetic fish were ovaries, thus suggesting that the sex-determining system of O. bidens is male heterogametic (XX-XY). The present study is the first to establish a procedure for inducing gynogenesis, we also identified the sex determination system (SD) of O. bidens, that will be possible to produce only male.