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Sperm and egg in vitro storage effects on artificial fertilization and hatching in common carp (Cyprinus carpio L.)
Abstract
This is the first report showing fertilization of short-term stored eggs (19 °C) with short-term stored diluted sperm (0–2 °C) in common carp (Cyprinus carpio). From 5 males, fresh (control) and 7-day stored sperm were diluted with common carp extender. After collection of eggs from nine females, the eggs were immediately pooled into three groups from females 1–3, 4–6 and 7–9, according to the eggs' quality based on visual criteria. They were placed in beakers, covered and incubated at 19 °C (for each pool, 10 g of eggs from each female were used). The 7-day short-term storage sperm was incubated at 20 °C for 20 min and pooled before being used for fertilization. The eggs were fertilized with fresh and short-term storage sperm after 1, 3 and 6 h post storage of eggs and activated with hatchery water and Perchec solution. Sperm motility and velocity (50–54% and 80–115 µm/s) of 7-day short-term storage sperm at 0–2 °C were ensured by additional incubation of sperm at 20 °C for 20 min before being activated. Principal component analysis showed that the sorting of eggs of different females into three groups according to sperm quality, fertilization ability and production of malformed larvae was successful. It is recommended that overripe common carp eggs are stored for a maximum of 1 h. A period of 3 h can be used for better quality eggs and exceptionally, up to 6 h for very high-quality eggs. Higher larvae malformation was found in the 6 h aged eggs together with fertilized short-term storage and fresh sperm. In all quality groups of eggs, the effect of different sperm, activation medium and egg storage time on the level of malformations was observed. The activation solution for carp did not show any positive fertilization and hatching effects.
... Recently, a bunch of studies have been conducted for in-vitro shortterm storage of common carp sperm to investigate different effects of the storage period, extender, and environmental conditions (Cejko et al., 2022;Dietrich et al., 2021;Cheng et al., 2022aCheng et al., , 2022bShaliutina-Kolešová and Nian, 2022;Cheng et al., 2023;Zhang et al., 2023;Linhart et al., 2023). The initial sperm quality depends on the sires' biological characteristics and health condition, hormonal stimulation methods, spawning season, and frequency of stripping (Alavi et al., 2008;Kowalski and Cejko, 2019). ...
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 24h 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.
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.
The biology of fish sperm, an important topic in the research of reproduction and fisheries from basic science to evolutionary and applied aspects, has implications for aquaculture management, fish breeding and biological conservation. The quality of spermatozoa plays a vital role in fish fertility, and directly affects the health and performance of offspring. Environmental factors that affect determinants of spermatozoa quality usually reflected in the DNA methylation pattern of the spermatozoa epigenome, which may change offspring's performance. The aim of the present study was to conduct a review on existing data about DNA methylation in fish spermatozoa as a biological tool for identifying the quality of offspring based on their phenotypes and performances. Furthermore, this study provides valuable knowledge from fundamental to applied sciences dealing with enhancement of breeding selection, fish reproduction and environmental adaptation. The review also describes the individual parts related to DNA methylation in fish, including overview of the methods which can be used to study DNA methylation, DNA methylation dynamics and epigenetic inheritance; identification of DNA methylation changes in sperm function in response to internal and external environment constraints, and potential relationships between DNA methylation and physiological regulation of spermatozoa quality determinants. Overall, the present study revealed that our knowledge about intergenerational inheritance of the performance and adaptability of fish through sperm DNA methylation is very limited, and no general conclusion could be approached from literature mostly due to non‐standardized experimental protocol or analytical tools.
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.
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.
Fish egg quality can be markedly influenced by the oocyte age after ovulation. In this study, we examined the duration of oocyte ageing in the zebrafish (Danio rerio) and whether prolonged ageing is associated with the incidence of ploidy anomalies in the resulting embryos. Oocytes were incubated in vitro for 6 h post-stripping (HPS) at 26 °C and fertilized at 2-h intervals. Meanwhile, for eggs fertilized immediately after stripping, the fertilization, embryo survival, and hatching rates started at ~80%; these rates decreased to 39%, 24%, and 16%, respectively, for oocytes that had been stored for 4 h (p ˂ 0.05), and there was an almost complete loss of egg viability at 6 HPS. Furthermore, almost 90% of the embryos derived from 6-h aged oocytes died prior to hatching, and all larvae originating from 4- and 6-h aged oocytes showed malformations. The proportion of ploidy abnormal embryos was significantly greater at 4 HPS (18.5%) than at either 0 or 2 HPS (4.7% and 8.8%, respectively). The results revealed that zebrafish oocytes retained their fertilization potential for up to 2 h after stripping at 26°C and indicated the contribution of post-ovulatory oocyte ageing in the occurrence of ploidy anomalies in the resulting embryos.
Common carp (Cyprinus carpio) is the fourth most-produced species in worldwide aquaculture. Significant efforts are invested in breeding and preservation of genetic integrity of this important species. However, maintaining a carp gene bank in situ can be demanding due to the large body size of the fish. Moreover, ex situ gene banking in common carp is as in other fish species limited to sperm cryopreservation. Recent progress in reproductive biotechnology in fishes has allowed the transfer of germ stem cells (gamete precursors) between individuals or even species. After maturation, the recipients are producing gametes of the donor. Surrogacy can serve as a valuable tool in germplasm banking to recover cryopreserved germ stem cells. Some unfavourable characteristics of the donor species such as long maturation time or large body size hampering its reproduction can be overcome by choosing a surrogate with more convenient characteristics. Efficient protocols for cryopreservation of common carp male and female germ stem cells have been recently developed in our laboratory. The next step has been to assess the potential of smaller goldfish (Carassius auratus) surrogates to produce donor-derived gametes of common carp after intraperitoneal transplantation of testicular cells. High transplantation success was achieved when 44% of the surviving goldfish produced pure donor-derived gametes of common carp at the age of 3 years giving rise to viable progeny. Donor-derived identity of the offspring was confirmed by genotyping and the production of a typical phenotype corresponding to the donor species. Reproductive performance of chimeras was similar to goldfish controls. Assessment of gamete characteristics showed that the size of donor-derived eggs is between control common carp and goldfish eggs. Interestingly , flagellum length in donor-derived spermatozoa was comparable to the common carp flagellum and significantly shorter than goldfish flagellum. Genotyping of the Y chromosome locus in chimeric goldfish provided a novel insight on the fate of transplanted cells, where only genotypic females were capable of producing eggs, while both genotypic males and females were capable of producing sperm. The present technology can be combined with cryopreservation procedures to ease needs for common carp breeds preservation and their recovery using many times smaller goldfish surrogates but also serve as a convenient model to study interspecific surrogacy in cyprinids.
There have been some arguments concerning supplementary feed (cereals) based common carp production in fishponds and water pollution, mostly in Central Europe. Using Czech Republic (top producer in EU) as a benchmark and combining data on nutrient digestibility of feedstuffs used combined with analyses of literature data, we have assessed – nutrient footprint (∼9.4–10.8 kg N ha−1, ∼2.7–3.2 kg P ha−1; 1.5–4 × < EU crop-livestock sectors); nutrient utilization efficiencies (NUEN ∼36%, NUEP ∼50%; 1.5–1.7 × > EU livestock average); autochthonous nutrient removal (∼8–9.2 kg N ha−1, 1.4–1.6 kg P ha−1); eco-cost burden (13–29 × ≪ positive services); eco-services (∼74.5–100.6 million € country−1; ∼2375 € ha−1) of carp production in Central Eastern European Region (CEER). Digestible nutrients offered by natural prey (7.9% N, 1% P on dry matter basis) to carp are ∼5–8 times higher than those provided by cereals and remains the key determinant for production. Despite this, 70–90% of nutrient footprint from feeding is contributed by cereals. Neutral footprint (∼374 kg ha−1) and exclusively natural (up to 300 kg ha−1) carp production intensities were identified, following which, commercial interest of carp farming may falter (costing intangible losses >56.5 million € in CEER), despite achieving ‘greener-goals’. Per production cycle, carp aquaculture in CEER fishponds offer at least 579 million € worth of services. Our results show that carp production in ponds have lesser nutrient burden than crop and livestock productions in EU. Existing management of fishponds ‘barely meet’ optimum P requirements of common carp and present production intensity should not be vilified as a pollution causing activity. Risks and solutions for achieving both environmental (minimized footprint) and aquaculture goals (uncompromised production) are discussed.
An isogenic line is a group of animals with an identical genotype, and its use is widely accepted for bioresearch standardization. Isogenic lines in fish can be generated within two generations via uniparental inheritance and can facilitate studies for which standardization and consistency are required. The availability and use of isogenic lines is limited, but isogenic lines in some fish species have been generated. Their power has been demonstrated in fields including human disease modelling, drug development, toxicology and also sequencing projects. The genetic basis of desired traits in aquaculture species can be identified using iso-genic lines, which, in turn, will help to improve fish culture. This review summarized knowledge regarding the present status of isogenic lines in fish including approaches for their generation and verification, as well as challenges and potential applications for basic research and aquaculture.
Oocyte ageing is the most important factor affecting egg quality of several fish species after ovulation. Oxidative stress has been proposed as the initiator of the oocyte ageing process in other vertebrates. To identify the role of oxidative stress and apoptosis on the progress of oocyte ageing in the common carp Cyprinus carpio, changes in the relative mRNA abundance of selected transcripts were examined. The possible alteration in the oxidation status of the oocytes during ageing was also studied. In addition, the activity of antioxidant enzymes during oocyte ageing was evaluated. Oocytes from 6 females were incubated in vivo for 14 hours post-ovulation (HPO) and in vitro for 10 hours post-stripping (HPS) at 20°C before fertilization. Hatching rates were over 65% up to 4–6 HPO, finally dropping to 1.3% at 12–14 HPO.Hatching rates were over 65% up to 4–6 HPO, finally dropping to 1.3% at 12–14 HPO. Hatching rates were more than 70% for the eggs stored in vitro up to 6 HPS and then decreased to 21.3% at 10 HPS. The results demonstrated no significant changes in the relative mRNA levels of oxidative stress-related genes or genes involved in the cell cycle during the progress of oocyte ageing in common carp. Additionally, the amount of TBARS and carbonyls did not change as time elapsed following ovulation. The apoptosis-related genes however, were significantly altered following the prolonged time interval between ovulation and fertilization. The lack of response of both activities of antioxidant enzymes and oxidation products during oocyte ageing strengthens the conclusion that oxidative stress is unlikely to be a main factor determining the progress of oocyte ageing in common carp. However, an increase in the mRNA abundance of apoptosis-related genes demonstrates that apoptotic pathway might be involved in the progress of oocyte ageing.
The common carp (Cyprinus carpio) is one of the longest-produced sweetwater fish species, the global production of which covers around 3.4% (4.4 million tons in 2015) of the world’s fish production and fisheries. Carp is the third most significant fish species of the world’s aquaculture production and 97.3% of its global production is originated from aquaculture. Furthermore, carp amounts to 8.3% of the world’s aquaculture fish production. Its feed technology is fish meal-independent and is mainly based on cereals. In Europe, the common carp is the most important fish species of aquaculture populated with extensive polyculture. The Czech Republic, Poland, Hungary, Germany and Croatia are among the biggest carp production countries of the European Union. The combined output of the TOP 3 carp production countries of the EU (CZ, PL, HU) amounts to 67.7% of the EU-28 (2015). Trade between EU countries is done primarily in the form of live fish and secondarily as fresh, primary processed carp.
The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n = 5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg−1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.
Knowledge of in vivo and in vitro storage of fish eggs can improve hatchery techniques as well as better broodstock management, which are the most important factors affecting synchronization of the artificial fertilisation and consequently the effectiveness of the entire operation. The duration of egg viability after ovulation and stripping has not yet been determined for the pike. A better understanding and refinement of egg storage methods are strongly desirable to maximize the efficacy of mass production in pike hatcheries.
In fish, delayed spawning in nature, delayed egg collection in capture and delayed fertilization after egg stripping can lead to oocyte ageing and over-ripening. Oocyte ageing is reported as one of the most important factors affecting the egg quality. During over-ripening, morphological, physiological, biochemical, histological, cellular, and molecular changes occur inside the eggs and ovarian fluid. These alterations can negatively affect the egg fertilization capacity and successive developmental stages. The time period during which eggs remain viable after ovulation or stripping has been reported from a few minutes to a few weeks depending on the fish species and the storage temperature. Although several biomarkers that characterize the over-ripening phenomenon have been defined, the reliability of these parameters in the field of aquaculture is controversial. For future researches, inhibition of oocyte ageing by antioxidants could be a valuable research topic. In the present article, the morphological, physiological, biochemical, histological, cellular, and molecular changes that occur during oocyte over-ripening under in vivo conditions and during in vitro storage are reviewed and possible reasons for the decrease in viability of eggs are presented. Species-specific changes in the egg viability and larval development are also considered.
Fish egg quality can be defined as the ability of the egg to be fertilized and subsequently develop into a normal embryo. Similarly, sperm quality can be defined as its ability to successfully fertilize an egg and subsequently allow the development of a normal embryo. In the wild or under aquaculture conditions, the quality of fish gametes can be highly variable and is under the influence of a significant number of external factors or broodstock management practices. For these reasons, the topic of gamete quality has received increasing attention. Despite the significant efforts made towards a better understanding of the factors involved in the control of gamete quality, the picture is far from being complete and the control of gamete quality remains an issue in the aquaculture industry. Some of the factors responsible for the observed variability of gamete quality remain largely unknown or poorly understood. In addition very little is known about the cellular and molecular mechanisms involved in the control of egg and sperm quality. In the present review, the molecular and cellular characteristics of fish gametes are presented with a special interest for the mechanisms that could participate in the regulation of gamete quality. Then, after defining egg and sperm quality, and how can it can be accurately estimated or predicted, we provide an overview of the main factors that can impact gamete quality in teleosts.
Egg quality can be defined as the egg ability to be fertilized and subsequently develop into a normal embryo. The ability to produce large numbers of high quality eggs “on demand” is an important issue for the development of aquaculture. Egg quality is deeply influenced by environmental factors and husbandry practices. Egg quality is a difficult to measure phenotype and is therefore not currently included in most selection programs.Automatic phenotyping of early embryonic success (i.e., mortalities and deformities) is a challenge. Our understanding of what makes a good egg remains incomplete despite the identification of key molecular mechanisms participating in egg quality. The identification of robust and generic (i.e., shared by evolutionary distant species) molecular markers of egg quality is a challenge for the future of aquaculture.
Mirror carp (Cyprinus carpio) semen was collected by hand stripping for short-term preservation. Two groups of pooled semen were diluted with one of two extenders; a control group was undiluted. The three groups were stored at 4°C for 72 h. During preservation, spermatozoa motility (%) and duration of spermatozoa movement (s) were determined every 12 h. Following preservation, fertilization was carried out using the dry fertilization technique at 1 × 105 spermatozoa/egg. The highest fertilization rates were 21±3.60% in May and 16.67±2.08% in June, both significantly higher than in the control. The highest hatching rates, 69.60±11.86% in May and 89.13±10.75% in June, did not significant differ from the control.
Genetic and physiological manipulations can be carried out easily in teleost fishes due to their external fertilization and embryogenesis. These procedures are performed in the early stages of ontogenesis to produce the required characteristic, which then leads to an altered phenotype. Two trends in effective modification of genotype of fishes have been developed: (a) manipulation of the genome by inducing gynogenetic and androgenetic reproduction and (b) manipulation of ploidy. In both of these modifications the sex ratio is modified by genome manipulation, allowing the establishment of monosex populations and inbred lines. Individuals of sterile populations produced by genome manipulation usually show better growing rate than their fertile mates. Moreover, sterilization substantially reduces the risk of acclimatization, when exogenous fish species are imported into different aquatic ecosystems. In the early stages of ontogenesis — before the sex-determining genes are switched on in the progenitors of germ cells — physiological processes can also be manipulated with remarkable effects on phenotype. Alteration of the sex of young fish populations by hormonal treatments is a well-known example. For some commercially important species this method can be applied very effectively and has become general in the practice of breeding, while there are still many unelucidated questions in other species, including common carp. Present methods could be rapidly improved by combination of certain genetic and physiological manipulations. The genetically identical individuals of fish clones have both experimental and practical importance. A large number of clones can be produced and sustained in carp using a combination of different manipulations. Insertion of recombinant DNA constructs into the genome of fish provides a new, efficient way of improving current stocks of farm fish. Methods of production, selection and characterization of transgenic individuals of fish, gene transfer experiments performed on and availability of presently cloned genes of farmed cyprinids with special emphasis on common carp as well as possible developments of the area in the future are discussed in this review. The experimental results produced by using these new techniques will increase our knowledge of the basic processes of fish biology. Together with traditional methods they will also hopefully contribute to the development of a more efficient and rapid carp breeding system in the future. Although application of these methods will obviously require efficient measures for protection of natural populations, overreaction resulting in regulations with prohibitive effects concerning the use of these procedures in laboratories should be avoided.
Conditions for short-term preservation of carp semen in the liquid state at 4°C were investigated in several experiments. Collected milt was spread in a 1-cm-thick layer in small flasks and exposed to the open air. In these conditions, the percentage of motile spermatozoa and the fertilizing capacity were high for 2 days and then rapidly decreased to zero after 6-8 days. The survival rate was markedly enhanced by the addition of antibiotics (50 μg/ml streptomycin+50 IU bipenicillin). After 8 days of storage, almost 100% of spermatozoa were motile and on day 16 the fertilization rate was reduced by only 20% (dilution 10-3). Loss of fertilizing ability was compensated by use of a larger amount of spermatozoa during insemination. A high between-male variability was observed in the sperm preservation ability, in particular beyond day 8. Sperm dilution and/or washing in various media (seminal fluid, saline solution) did not improve preservation. Incubation under oxygen slightly improved the survival rate up to 6 days of storage. Spermatozoa underwent morphological changes during storage, e.g. winding of flagella, loss of the central doublet, appearance of verrucosities on the sperm head accompanied by the detachment of the nuclear envelope and the plasma membrane from the nucleus. There were 3% abnormal spermatozoa in fresh sperm and 46 and 64%, respectively, after 6 and 15 days. The mortality rate of larvae issued from eggs fertilized by spermatozoa stored for 15 days was not different from that of the controls (fresh sperm) and there were no developmental abnormalities.
One of the major objectives of the aquaculture industry is the production of a large number of viable eggs with high survival. Major achievements have been made in recent years in improving protocols for higher efficiency of egg production and viability of progeny. Main gaps remain, however, in understanding the dynamic processes associated with oogenesis, the formation of an egg, from the time that germ cells turn into oogonia, until the release of ova during spawning in teleosts. Recent studies on primordial germ-cells, yolk protein precursors and their processing within the developing oocyte, the deposition of vitamins in eggs, structure and function of egg envelopes and oocyte maturation processes, further reveal the complexity of oogenesis. Moreover, numerous circulating endocrine and locally-acting paracrine and autocrine factors regulate the various stages of oocyte development and maturation. Though it is clear that the major regulators during vitellogenesis and oocyte maturation are the pituitary gonadotropins (LH and FSH) and sex steroids, the picture emerging from recent studies is of complex hormonal cross-talk at all stages between the developing oocyte and its surrounding follicle layers to ensure coordination of the various processes that are involved in the production of a fertilizable egg. In this review we aim at highlighting recent advances on teleost fish oocyte differentiation, maturation and ovulation, including those involved in the degeneration and reabsorption of ovarian follicles (atresia). The role of blood-borne and local ovarian factors in the regulation of the key steps of development reveal new aspects associated with egg formation.
This study was designed to optimize a short-term storage protocol for common carp sperm at 4 oC under aerobic condition. Sperm from individual males were collected directly with or without extenders. The results demonstrated that in general, it was similar effect to collect sperm directly in extenders and keeping sperm for 0.5 h after collection without extenders. Sperm was diluted with eight selected extenders (sperm: extender = 2:1, 1:1 and 1:9) and undiluted sperm was used as a control. Sperm and seminal plasma parameters (sperm motility, velocity, membrane integrity, sperm concentration, osmolality and pH in seminal plasma) were evaluated in sperm stored on ice under aerobic conditions at 0, 2, 4, 6 and 8 days post stripping (DPS) using the computer- assisted sperm analysis system. Results showed that 1:1 and 2:1 dilution maintained higher sperm function and more sperm for a longer period. After 8 DPS, the best sperm quality and quantity was recorded in the common carp seminal plasma supplemented with 50 mM NaCl, Cejko extender (2 mM CaCl2, 1 mM MgSO4, 20 mM Tris, 110 mM NaCl, 40 mM KCl, pH 7.5 and 310 mOsm/kg) supplemented with/without 25 mM KCl/NaCl. The reduction of spermatozoa number with time during short-term storage but varied according to different dilution ratios and extenders. At 8 DPS, sperm count has dropped by 22.9% in a dilution of 1:1 compared to 50.3% in sperm without dilution. Extenders with diluted 1:1, especially Cejko solution, largely postponed sperm reduction, 21.3% compared to 55.5% for sperm stored without extenders.
The purpose of this study was to investigate the effect of an activation solution (AS) (hatchery water (pH 7.9 and 4 mOsm/kg), Perchec solution (5 mM KCl, 45 mM NaCl, 30 mM Tris-HCl; pH 8.0 and 130 mOsm/kg) and Cheng solution (40 mM NaCl, 20 mM Tris; pH 8.0 and 100 mOsm/kg) on aging sperm of common carp (Cyprinus carpio) after storage in a carp artificial seminal plasma (carp ASP: 2 mM CaCl2, 1 mM MgSO4, 40 mM KCl, 110 mM NaCl, 20 mM Tris; pH 7.5 and 310 mOsm/kg) for 7 days without antibiotics at 4 °C. Fresh milt and undiluted aging milt were used as the control groups. Milt (5 ml) was collected and diluted 1:1 in a carp ASP in 50 ml containers and stored at 4 °C under aerobic conditions. The highest sperm motility was found 45% and 36% at 15 s and 45 s post sperm activation (PSA) in hatchery water. Additionally, the curvilinear velocity (VCL, μm/s) and straight-line velocity (VSL, µm/s) of sperm were the highest in hatchery water and the solution with 5 mM KCl, 45 mM NaCl, 30 mM Tris and pH 8.0, which were above 122 µm/s and 87 µm/s at 15 PSA. Sperm concentration in aging sperm of common carp is known to decrease rapidly, so prior to fertilization, sperm concentration was determined and accurately set to 200,000 sperm per egg. The fertilization of eggs and hatching rate after 7 days of sperm storage in carp ASP were 85–87% and 78–81% in both ASs of hatchery water and Perchec solution. Less than 1% sperm motility, fertilization and hatching rate were observed in undiluted sperm (control). In conclusion, the fertilization ability of aging common carp sperm in carp ASP within different ASs was confirmed for practical use in aquaculture.
Methods of induction of uniparental inheritance have been developed for model organisms as well as for species of commercial interest to fix sex-specific traits in breeding programs or to restore breeds or lines from cryopreserved sperm in aquaculture important species. Androgenesis in common carp (Cyprinus carpio) has been successfully induced by egg nucleus inactivation with γ-, X- or UV-ray irradiation but these techniques are not widely applicable for laboratory or commercial use on practical, financial and safety grounds. In the past decade a promising low-cost, low-tech cold-shock approach for successfully inducing androgenesis has been demonstrated for loach, Japanese flounder and zebrafish. The aim of the current study was to develop a cold shock methodology for application in common carp, a freshwater species of high commercial interest and a widely used model species. Gametes were collected from wild-type females (dominant green phenotype) and Koi males (recessive blonde phenotype) to enable easy identification of successful induction of androgenetic haploid progeny. A combination of different temperature treatments (0, 2, 4, 6, 8 °C) and different cold-shock durations (15, 30, 45, 60, 75 min) applied shortly after gamete activation (3 s after fertilization) were initially trialed. Optimal condition for egg nucleus elimination was a cold-shock at 2 °C for 60 min duration where hatching rate of haploid progeny reached 35.6% and 26.26 ± 10.19% across two attempts. Double haploid induction was then attempted with three replicates (300 g egg) with following parameters 2 °C cold-shock for 60 min, then transferred into 20 °C and subsequently treated by a heat shock arresting first mitotic cleavage applied 40 min at 40 °C for 2 min. These combined treatments resulted in reduced fertilization and hatching rates for all replicates and low yield of progeny (1.09–1.28% in experimental incubation, <1% in hatchery incubation). A genome wide SNP analysis of a subset of progeny confirmed that they were double haploids. Cold-shock androgenesis was found to be effective in common carp, providing a new possibility of uniparental inheritance induction for isogenic line production.
We previously demonstrated that artificial seminal plasma (ASP), developed for common carp (Cyprinus carpio), enabled considerable improvement of the fertilizing capacity of sperm in this species. Here, for the first time, the effectiveness of using ASP (ASP: 2 mM CaCl2, 1 mM MgSO4, 40 mM KCl, 100 mM NaCl, 20 mM Tris; pH 7.5 and 310 mOsm kg⁻¹) for short-term storage of tenfold diluted sperm for up to two weeks at 10 °C was evaluated. Additionally, the fertilizing capacity of stored sperm was investigated. Sperm were stored in a volume of 5 ml in containers equipped with ventilation plugs enabling gas exchange of the stored sperm. During the study, the quality of the sperm was verified using the computer-assisted sperm analysis (CASA) system after 1, 2, 5, 9, 11 and 14 days of storage. After 14 days of common carp sperm storage in ASP sperm motility (MOT, %), the progressive motile sperm (PRG, %) and curvilinear velocity of sperm (VCL, μm s⁻¹) were confirmed to be 55%, 25% and 120 μm s⁻¹, respectively. At the same time, in undiluted sperm (Control), we did not observe sperm motility. Only until the 2nd day of storage in undiluted samples motility and sperm kinetic parameters remained high level (84%, 34% and 144 μm s⁻¹ for MOT, PRG and VCL, respectively). The fertilization of eggs and hatching rate after 14 days of sperm storage in ASP were 80% and 40%, respectively, and did not differ significantly from the fertilization and hatching rate determined after fresh sperm was used for fertilization. In summary, for the first time, the fertilization capacity of common carp sperm after 14 days of short-term storage under ASP conditions was confirmed, indicating the possibility of using previously collected and diluted ASP sperm in reproduction at a time convenient for the breeder. Thus, it is possible to reduce the stressful and time-consuming handling of fish during breeding under controlled conditions, which has an impact on the welfare of spawners.
Koi herpesvirus disease (KHVD) is currently the most serious threat to global carp farming. Prevention is a sensible strategy for tackling this disease and improved genetic resistance of carp strains is a desirable breeding goal. To study the potential for multitrait selection, the objective of the current study was to estimate the genetic correlations between KHVD resistance and production traits in Amur mirror carp. A total of 1500 fingerlings from four factorial crosses of five dams and ten sires were challenged with Koi herpesvirus (KHV). Juvenile growth-related traits were collected on the same individuals before the challenge test. Production traits were measured on siblings of the challenged population at different life stages (yearling to market size). The estimated heritability for resistance to KHVD was 0.43 ± 0.08 on the observed scale and 0.72 ± 0.13 on the underlying liability scale. Most genetic correlations between KHVD resistance and important production traits were insignificant, showing that selection for improved production traits would not increase susceptibility to KHV and vice versa. However, resistance to KHVD was negatively correlated with Fulton's condition factor (FC) after the second overwintering and relative head length (RHL), relative body height (RBH) and relative body width (RBW) from the second growing season to the market size, with a more prolonged body shape of Amur mirror carp (genes from Amur wild scaly carp, Cyprinus rubrofuscus) being associated with higher KHVD resistance. Intermediate favorable genetic correlations between KHVD resistance and log-log residuals of headless carcass yield (0.37 ± 0.14) and fillet yield (0.44 ± 0.13) at market size suggested that selection for improved yields of edible body parts might indirectly lead to a slight improvement in KHVD resistance and vice versa.
Storage of refrigerated semen is a simple and inexpensive procedure that can facilitate the management and reproduction programmes in aquaculture and allows to store semen for in vitro reproduction, quality analyses, hatchery production support, selective in vitro breeding, disease diagnosis, transportation, cryopreservation and advanced molecular studies. Implementation of semen short‐term storage protocols for threatened or endangered fish represents a useful strategy for preservation of these species. Semen cold storage as technique is a useful strategy for the conservation of fish sperm. Analysing the male broodstock sperm quality during cold storage is a tool to identify the reproductive ability of cultivated fish. Thus, in this review we analysed the most relevant factors that affect sperm quality during cold storage of male broodstock semen as well as its effects on motility, viability, mitochondrial membrane potential, superoxide anion level and DNA fragmentation.
Fish sperm quality assessment is helpful for optimizing production and for monitoring the environmental state. Sperm can be monitored relatively easy and, to date, various analyses have been applied and proven to be helpful in this task. Among them, sperm motility parameters such as sperm speed are one of the main performance traits during assisted fish reproduction. Apart from motility the sperm concentration, volume, and seminal plasma pH and osmolality are also frequently evaluated and are the main sperm quality indicators measured in fish sperm. However, other parameters also determine sperm fertilization potential. Recent knowledge reveals several additional parameters of high importance for sperm function. Among them are DNA integration, membrane stability, mitochondria status and enzymatic activity. Measuring all these parameters in fish sperm provides complex knowledge regarding male fertility and helps to improve broodstock maintenance protocols as well as gamete handling and fertilization processes. This review focuses on the presentation of the sperm quality measures for
freshwater and marine species of the fish and provides information regarding recent methods of sperm quality evaluation.
The quality of fish sperm is characterized by relatively high individual variation. Moreover, under artificial conditions, the collected sperm loses its motility and viability very rapidly, which may decrease fertilisation success. Therefore, techniques able to secure and maintain a high biological value of collected fish sperm are needed in hatchery practice. Herein, we used previously composed artificial seminal plasma (ASP) containing 2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris, 110 mM NaCl and 40 mM KCl (pH 7.5 and 310 mOsm kg−1) to dilute common carp sperm samples for 1 h storage prior to fertilisation. Sperm was collected from mature males and, after checking its motility (MOT) using the CASA system, was divided into high (range 55–65%) and low (range 5–10%) quality. Samples of both sperm qualities (high/low) were diluted tenfold (1: 9; sperm: extender) in ASP and stored for 24 h at 10 °C. Motility (MOT, PRG) and velocity (VCL, VSL) of low- and high-quality sperm diluted in ASP increased after 1 h of storage compared to undiluted sperm. Moreover, the fertilisation capacity of low-quality sperm (previously diluted and stored for 1 h in ASP) increased three times compared to undiluted sperm of the same quality. Prolonging the time of sperm storage in ASP to 24 h results in the further increase of MOT, PRG and VCL in the low quality samples and PRG in the high quality samples. The results of the presented study confirm the possibility of common carp sperm revitalisation using ASP. The utilisation of such sperm in hatchery practice is possible since the fertilisation capacity of sperm, previously diluted and stored for 1 h in ASP, was over 90% regardless of the initial sperm quality (high/low).
It is essential to define an optimized standard method to assess the fish sperm quality to minimize the differences between the results obtained by different laboratories. Only this optimization and standardization can make them useful from academia to industry. This study presents the validation of sperm motility assessment using a CASA-Mot system for three endangered diadromous fish species: European eel (Anguilla anguilla), Atlantic salmon (Salmo salar) and Siberian sturgeon (Acipenser baerii). To attain this goal, different technical and data processing methods were tested: 1) magnification lens (×10 and ×20), 2) Spermtrack® reusable chambers (10 and 20 μm depth) and 3) different frame rates (50 ≥ FR ≤ 250). The results suggested that the sperm motility assessment for eel, salmon and sturgeon should be performed at 200, 250 and 225 frames s⁻¹, respectively. Moreover, to obtain a high number of analysed spermatozoa in less time and a natural movement of the sperm cells, it is recommended to use ×10 objective and 20 μm depth. In conclusion, different technical settings influence sperm kinetic parameters and should be validated for each fish species to allow the comparison of results between laboratories.
In this review we provide an overview of the components of the spermatozoa playing an important role in reproductive success beyond fertilization, showing the relationship between the integrity of the diverse elements and the development of a healthy offspring. The present knowledge about fish sperm chromatin organization, epigenetic modifications of DNA and histones and sperm-borne RNAs, essential in controlling embryo development, is summarized, pointing out the possibility of using specific genes or transcripts as biomarkers of sperm quality. Data about commercial species are reported when available and more detailed information about zebrafish sperm is presented.
The short-term storage of salmonid semen is a viable method for in vitro fertilisation. Previous studies have found that short-term storage affects sperm motility, compromising quality and fertilising capacity. However, the functional characteristics of the spermatozoa of O. mykiss during storage time and its relation to the spawning period are little known. This study was designed to evaluate the effects of in vitro short-term storage on sperm functional parameters in O. mykiss, determined by flow cytometry. Semen samples of the first spawning – undiluted (SSD) and diluted (SD) (Storfish® 1 : 2v/v; IMV AI solutions, France) – were stored at 4 °C for 14 days. Motility, viability (PMI: plasma membrane integrity) and mitochondrial membrane potential (ΔΨM) were assessed. On the fifth day of storage, spermatozoa showed a motility >70% (SSD: 78.3% versus SD 85.0%), PMI (81.5% SSD/87.2% SD) and ΔΨM (72.5% SSD/SD 80.0%) (P < 0.05). However, a significant decline in the percentage of all functional parameters (P < 0.05) was observed after 5 days of storage for all samples of both undiluted (SSD) and diluted semen. In conclusion, the results here provide new data on O. mykiss sperm quality with respect to in vitro short-term storage evaluated by flow cytometry.
In this study, a short-term storage of pre-activated eggs of the Japanese ornamental (koi) carp, Cyprinus carpio L., at three different temperature regimes is reported. Koi eggs were stored at low temperatures (6-9oC), at variable or high temperatures (12-31C) and at moderate-stable temperatures (20-24.5oC). Survival of the developing embryos was examined at the first and the second day post-activation, and in hatch-out larvae. Survival was calculated for each treatment by linear regression Y = a-bX (P± 0.01), except for eggs incubated at temperature of about 20 C(P > 0.1).The specific mortality index (b/a 100), as a ratio between the mortality rate (b) and the fertilization rate (a), indicated that the highest mortality is associated with the high and unstable storage temperatures, while the lowest mortality is with the moderate and stable storage temperatures. Eggs can be stored at moderate and stable temperatures for a maximal duration of 6h, yielding survival of hatch-out larvae higher than 50%. In two trials, fertilization potency of sperm stored in a domestic refrigerator (5-9oC) for 5h, was compared with freshly stripped sperm and no differences in fertilization rates were found between the two treatments.
Paleogeographical, morphological, ecological, physiological, linguistic, archeological and historical evidence is used to explain the origin and history of the domestication of the wild carp. The wild ancestor of the common carp originated in the Black, Caspian and Aral sea drainages and dispersed east into Siberia and China and west as far as the Danube River. It is represented today by the uncertain east Asian subspecies Cyprinus carpio haematopterus and by the east European Cyprinus carpio carpio. There is evidence that the Romans were the first to culture carp collected from the Danube, and that the tradition of the “piscinae” was continued in monasteries throughout the Middle Ages. Distribution of the carp west of the Danube's piedmont zone was clearly caused by humans, as was the introduction throughout the continents. Some domestication in China may have been independent of similar activities in Europe, but most of the modern-day activities with the common carp in far east Asia are restricted to the domesticated carp imported from Europe, or at best to hybrids of local and imported strains. The xanthic (red) common carp seem to have first appeared in early cultures of Europe, China and Japan but reached their fame through recent artificial selection of multicolored aberrants in the Niigata Prefecture of Japan. The production of the colored carp — the Japanese “nishikigoi” — presently exceeds in monetary value the production of carp as human food. The nishikigoi as “swimming flowers” delight modern people as much as the taste of carp delighted the Romans at the beginning of carp domestication.
The spermatozoa of various marine teleosts (sea bass, Dicentrarchus labrax; sea bream, Sparus auratus) or fresh water teleosts (trout, Salmo gairdneri; pike, Esox lucius; guppy, Poecilia reticulata) were diluted in media of different salinities. Motility, morphological changes and fertilizing ability were the criteria used in judging the effects of such treatments. The medium best adapted to dilution of sea-fish sperm had a salinity of about 20‰. Sperm motility was increased and prolonged, and fertilization rate was significantly improved (P<0.05) for the sea bass at a 1 1000 dilution. For freshwater fish (trout and pike), an extender with about 7‰ salinity increased motility time and fertility ability (P<0.01) as compared to freshwater. After dilution in fresh water the structure of trout spermatozoa was considerably altered (rupture of plasma membrane and mitochondrial swelling). When spermatozoa were diluted in the extender, there were no significant structural changes in trout, but alteration occurred in the mid-piece of the guppy spermatozoon. It is concluded that fresh water or sea water are not the best media for the practice of artificial insemination in freshwater or marine fish. Investigations should be carried out to define the best extender for use in techniques of artificial insemination in fish produced in aquaculture.
Short-term storage of the eggs of rainbow trout Oncorhynchus mykiss, brown trout Salmo trutta, and grayling Thymallus thymallus was investigated to examine factors that limited egg viability and to compare storage with and without ovarian fluid. Egg viability was similar after 2 h, but after 4 h, egg fertilization rate was lower by 14-26% for eggs stored in Ovarian fluid and lower by 23-51% in eggs stored without ovarian fluid. Substitution of a buffered physiological saline solution for ovarian fluid was tested for brown trout and grayling but did not improve egg viability. The wet weight of eggs during storage without ovarian fluid decreased significantly. Water influx during hardening was higher for eggs stored without ovarian fluid than in controls and eggs stored with ovarian fluid. However, water loss due to storage could not be completely compensated, so the wet weight of hardened eggs that had been stored without ovarian fluid remained lower. Ovarian fluid pH and activities of protease during egg storage with ovarian fluid significantly increased, and peptide levels decreased.
The objective of the study was to compare carp sperm motility performances (sperm velocity and motility rates) from 10 males including fertilizing ability (hatching rates from 10 males and eight females) as a function of time elapsed after sperm exposure to activation medium in two situations: firstly activated sperm and sperm which had terminated swimming and was ‘re-activated’ after incubation in a K+ rich (200 mm KCl) non-swimming solution. In case of both initial (first) and secondly activated spermatozoa, the motility was triggered in hatchery solution (HAS, 11.2 mOsmol) and in carp activation solution (CAS, 128.9 mOsmol) containing 45 mm NaCl, 5 mm KCl, 30 mm Tris–HCl while also adjusted to a pH of 8.0. First time activated sperm showed significantly higher relative motility, sperm velocity and fertilizing ability compared to re-activated sperm. The carp spermatozoa (in either first or second activation) rapidly lost their fertilizing ability as a function of exposure time of sperm to diluents prior to addition to eggs: this shows that spermatozoa must be in contact with eggs as soon as their motility is triggered. When sperm was firstly activated in CAS and also activated a second time in CAS (labeled CASCAS) the hatching rate was significantly higher at egg contact after 10, 20, 30, and 120 s of activation. Also at 20 s after the second activation of the sperm higher sperm motility was observed compared to the first activation. This study showed that incubation of spermatozoa in a K+-rich incubation medium can mitigate the affects of structural damages occurring in re-activated sperm, which may help spermatozoa to increase their motility and fertilization. To our knowledge, the results presented in this study document for the first time that fertilization can be achieved with sperm re-activated a second time while being exposed to a incubation medium that permits ATP reloading within the flagellum. Previous studies have show the potential for recovery of motility, however, the effect on possible fertilization is hitherto unknown. It critical outcome of the study clearly indicated the need for avoiding the use of different, subsequent activation media (e.g. first and second activation) but only on the same medium for both steps (see above CASCAS).
A computer-aided semen analysis system was used to assess the % motile cells following storage of carp semen in 11 different buffers at 2, 5 or 22° C. BWW and TLP were the most suitable storage buffers because carp semen stored at 5° C in these buffers following activation showed no significant decrease in % motile spermatozoa up to 24 h. But, in most of the other buffers (Fish Ringer, Cytomix, Cortland, FRT, Mannitol, FPS, NAS and TSM) the motility potential was lost by 2 h. Storage was best at pH 6–9 and at 5° C. Carp spermatozoa exhibit three distinct motility patterns, namely ‘linear’, ‘circular’ and ‘haphazard’, the proportion of spermatozoa with a particular motility pattern depending on storage buffer and time. All spermatozoa with a linear trajectory had high VSL, STR and LIN; those moving in circles had low VSL, STR, LIN and BCF and those with a haphazard trajectory were distinct in that they had the highest ALH and their VSL, STR, LIN and BCF were higher than the circular moving spermatozoa and lower than the spermatozoa exhibiting linear trajectory. The study also demonstrates a pronounced time-dependent decrease in VCL, VAP, VSL and ALH of carp spermatozoa following activation with water or low osmolality solutions. This study provides for the first time data related to seven motility parameters of carp spermatozoa and demonstrates how these parameter values could be used to evaluate quality of carp milt following storage in different buffers. It confirms that carp spermatozoa exhibit linear or circular trajectories and provides evidence for a third type of trajectory described as haphazard. All three motility patterns could be discriminated objectively on the seven motility parameters.
This paper is a review of the literature on eggs of carp (Cyprinus carpio L.) and some related species and includes data taken from the former USSR and eastern European countries. Reported data relate to fecundity, egg morphology and composition, and fertilization. Information is also available on the ovarian fluid composition with characterization of proteins and lipids. The fecundity of common carp is very high, ranging from 100 000 to 300 000 eggs · kg−1 body weight per oogenetic cycle, with one cycle/year in the case of females reared in an outside natural pond environment. The diameter and weight of the eggs are in the range of 1.24–1.42 mm and 0.86–1.41 mg, respectively. The ooplasm includes large amounts of yolk and various organelles (endoplasmic reticulum, Golgi apparatus, mitochondria), cortical granules and alveolae. The main characteristics of cyprinid ovarian fluid are an osmotic pressure of about 300 mOsm, a Mg2+ concentration of 2.58 mM and a high ovarian fluid pH of 9. Energy in the form of ATP which is necessary for egg metabolism originates from glycolytic and oxidative reactions. The egg has a relatively thick vitelline envelope (VE) or zona radiata which is reorganized into the fertilization envelope (FE) after fertilization. The micropyle located at the animal pole is a funnel-shaped structure leading spermatozoa to the ooplasmic surface on which a fertilization cone develops after fertilization. A site of sperm attachment is identified on the plasma membrane at the level of the internal aperture of the micropylar canal. Major differences are observed between the two outermost VE and FE layers, as revealed by electron microscopy, enzyme or carbohydrate cytochemistry, and imunohistochemistry. FE extracts have strong bactericidal and fungicidal effects.
Although the common carp, Cyprinus carpio, has been cultivated for several thousand years and is produced in large quantities, research on reproduction has been very limited. Traditionally, spawning occurred naturally in situ in rearing ponds. In slightly improved methods large breeding ponds stocked with brood fish were devoted to reproduction with fry collection in autumn, or in small spawning ponds with collection of larvae a few days after hatching. Controlled reproduction in hatcheries started only in the 1950s. This paper reviews some of the basic work on carp sperm and describes the technologies of artificial reproduction. The sperm is of a primitive type with uncondensed chromatin and a small midpiece. As in most teleost fish, the sperm is immotile in the male genital tract and in the semen and is activated after release into fresh water. The initiation of motility is due to the decrease in osmotic pressure. The structure of the flagellum is rapidly disorganized in fresh water and sperm stop moving after 30 s. When dilution occurs in a 50 mM NaCl solution, the osmotic change is sufficient to initiate motility, but the flagellum is not disorganized and swimming lasts a few minutes. Motility depends mainly on endogenous ATP stores, about spermatozoa, and stops when 50–80% of the ATP is exhausted by hydrolysis. The procedure of artificial insemination includes collection of gametes (from “hypophysised” males and females), mixing in an extender (45 mM NaCl, 5 mM KCl, Tris 2.5 mM, glycine 19 mM, pH 8) in a ratio 1 litre of eggs to 1 litre of diluent and 1 ml of semen. Sperm motility can be triggered during collection by contamination of the semen with urine; this could interfere with fertilization and, if present, urine should be discarded by pouring out the top part of the tube. The sticky layer of the egg is removed by adding a “dissolving solution” (20 g urea/1 + 4g NaCl/1 of water) or milk (diluted in water) to the fertilized egg and stirring. One hour later the swollen eggs are transferred to incubators (usually Zug bottles, sometimes 200 litre circular jars).
About 70% of eggs of Cyprinus carpio reached the eyed stage in fertility tests with 1 ml of cryopreserved semen added to 1 g of eggs. When 1 ml of semen was added to 100 g of eggs, few eggs reached the eyed stage. In two fertility tests with sperm preserved for 342 days, using 5 g of eggs inseminated with 1 ml of semen, percentages of eyed eggs were 31.5 and 25.5, respectively. In a series of fertility tests, the chemical constituency of the medium in which fertilization was attempted influenced the fertility of cryopreserved sperm.
Common carp is one of the leading species in world aquaculture, but selective breeding for growth rate has not been actively pursued in this species after unsuccessful selection experiments. We estimated heritability for growth-related traits at 8 weeks of age in Hungarian Synthetic Mirror carp at Vodnany (Czech Republic). Parentage assignment with eight microsatellite markers was used in a full factorial cross of 10 dams×24 sires. Out of 550 offspring, 95.3% could be assigned to a single parental pair. Animal model heritability estimates were 0.33±0.08 for weight 0.33±0.07 for length and 0.37±0.07 for Fulton's condition factor (K). Maternal effects and dominance were not significantly different from zero. The genetic correlation between weight and length was 0.98, and negative correlations were found between K and length (−0.38) and K and weight (−0.17). It is concluded that selective breeding for increased weight gain can be successful in juvenile carp, using indirect selection for length. However, the facts that heritability was estimated at 8 weeks of age and not at harvest weight, as well as a possible amplification of additive variance by competition, are limitations to the applicability of the present results.
Survival of Silurus glanis ovulated oocytes (oval measured by the capacity of normal development i.e. percentage of normal and abnormal hatched larvae after in vivo and in vitro storage and exposure to sperm activating or immobilizing solutions and urine was studied. In the case of ovulated oocytes left in the ovaries, total hatching and abnormal hatched larvae (in % of hatching) were respectively 74 and 8.2% immediately after ovulation, 77 and 8.6% after 2 h, 54 and 18% after 4 h, and 38 and 50% after 6 h of storage. Four and 6 h stay of ovulated oocytes in ovaries resulted in a significant increase of abnormal hatched larvae (p<0.01). For the ova stored in vitro, the capacity to undertake a full embryonic development after fertilization was not significantly changed either after 8.5 h at 19 degrees C or 3.5 h at 25 degrees C; there was no ova survival at all after 3.5 h storage at 8 degrees C, 12 h at 19 degrees C and 8.5 h at 25 degrees C. There was a significant increase of abnormal larvae after 3.5 h at 25 degrees C (74%, p<0.001) and after 8.5 h at 19 degrees C (37%, p<0.05). Exposure of non inseminated ova to water buffered to pH 7.0 (5 mM Tris-HCl) to sperm activating solution (17 or 41 mM NaCl, 5 mM Tris-HCl, pH 8.0) or to immobilizing solution (200 mM NaCl, 30 mM Tris-HCl, pH 7.0) resulted in a regular and rapid decrease of their capacity of development; this was 10% of normal hatched larvae within 4 min in water, and within 6-8 min in the NaCl solutions. A similar situation was observed after exposure to urine with a loss of embryonic development of 30% within 3 min. These results indicate that all steps of the whole procedure of gamete collection and artificial insemination should be carried out rapidly as soon as possible after ovulation.
The effect of different parameters on short-term storage capacity of turbot ova was assessed over a 45-h period after ova collection for fertilization rates and over a 9-h period after ova collection for hatching rates. Increasing the volume of ova sampling from 0.5 to 2.5 mL, as well as adding an antibiotic-antimicotic solution or oxygen did not significantly change the storage capacity of ova. Regarding the hatching rates, a higher storage ability was recorded at 8 and 13°C, compared to 3°C. The mean composition of the ovarian fluid was determined (n = 57 spawns). Use of a diluent mimicking the ovarian fluid significantly decreased the storage ability as assessed by the fertilization rates but did not modify the hatching rates. Diluting ova in an artificial ovarian fluid deprived of calcium significantly decreased the fertilization and hatching rates during the storage period. Furthermore, addition or not of soybean trypsin inhibitor (Sigma T 9003) to the artificial ovarian fluid deprived of calcium did not significantly change the results. Storage capacity of control batches of ova was low: at 13 °C, without any diluent and when ova were fertilized 3 h after stripping, the hatching rate was lowered to 62.4± 29.4 % (mean ± SD) of the initial value.
Carp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3.000-fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145-1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation-reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially "poor" quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2 degrees C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation-reactivation occurred in ATP-Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year-round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.
Studies on the flagellar movement of carp spermatozoa induced by dilution in distilled water allowed us to describe a sequence of early, rapid morphological and kinetic changes which begin at the very tip of the flagellum. They cause the progressive folding of the axoneme which ends stuck to the head within 90-120 seconds after the initiation of motility. However, the axonemal machinery remains functional as the folding can be reversed after transfer back into a high osmolality medium and partially folded flagella were able to propagate efficient waves along the non-folded proximal portion of the axoneme. The data also revealed that the membrane area of the terminal piece exhibits strong sensitivity to hypotonicity. These results suggest that in the normal freshwater medium, the brief swimming period allowing fertilization of oocytes is limited by the osmotic stress induced coiling of the carp sperm tail and not by ATP stores.