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International audience; The aim of this study was to investigate the respective contribution of maternally-inherited mRNAs and proteins to egg molecular cargo and to its developmental competence in fish using pikeperch as a model. Our study provides novel insights into the understanding of type-specific roles of maternally-inherited molecules in fish. Here we show, for the first time, that transcripts and proteins have distinct, yet complementary, functions in the egg of teleost fish. Maternally-inherited mRNAs would shape embryo neurodevelopment, while maternally-inherited proteins would rather be responsible for protecting the embryo against pathogens. Additionally, we observed that processes directly preceding ovulation may considerably affect the reproductive success by modifying expression level of genes crucial for proper embryonic development, being novel fish egg quality markers (e.g., smarca4 or h3f3a). These results are of major importance for understanding the influence of external factors on reproductive fitness in both captive and wild-type fish species.

International audience; This study aimed to assess the effect of constant darkness applied to fish during controlled breeding on reproductive traits in domesticated females of Eurasian perch. Based on the assumption that keeping fish in constant darkness during the reproduction operation may reduce stress, suspected to be responsible for variable spawning effectiveness in this species. Two conditions were assessed (16 h light per day [group 16L] and constant darkness [group 0L], two tank replicates per condition). The reproductive protocol involved a 7-day-long adaptation period for group 0L where photoperiod was reduced by 2.3 h a day down to constant darkness. After the adaptation period, two hormone injections (salmon gonadoliberin analogue) were applied to both groups: priming (10 mu g/kg) and resolving (25 mu g/kg) with a 7-day interval between them. During the study, morphometric indices were recorded and blood, brain, and pituitary samples were collected to assess stress markers and determine hypothalamic-pituitary-gonadal axis functioning via measuring blood plasma hormones, as well as gonadoliberin and gonadotropins (luteinising hormone [LH] and follicle-stimulating hormone [FSH]) transcript abundance (n = 7 for each group at each sampling point). In addition, kinetics of the final oocyte maturation (FOM) process, ovulation rate, and egg quality of each group was monitored (n = 12 for each group). The results indicated that there were no differences in terms of morphometry, FOM kinetics, and most stress indices between groups throughout the experiment, except haematocrit, which increased immediately following the acclimation period in fish kept in darkness. Constant darkness negatively affected plasma levels of 17 alpha,20 beta-dihy droxy-4-pregnen-3-one (DHP) and LH transcript expression at the time of the second hormone injection. This indicated that exposure to constant darkness negatively affected priming of the hormonal dose applied, resulted in the disruption of ovulation, and reduced ovulation rates (50%) for group 0L, as compared to 16L (91%). The findings of this study clearly indicate that constant darkness may have significant deleterious effects on reproductive traits throughout out-of-season induced, hormonally supported, controlled reproduction. Therefore, we advise against the use of constant darkness when managing broodstock reproduction in domesticated Eurasian perch.

BackgroundIn Metazoans, embryonic development relies on maternally-inherited mRNAs and proteins that are critical for early developmental success and known to play major roles at later stages, beyond zygotic genome activation. However, very poor concordance between transcript and protein levels in oocytes and embryos of vertebrates suggest that maternally-inherited proteins and maternally-inherited mRNAs are playing different roles in unfertilized eggs, not considered to date comprehensively. The aim of this study was to investigate the respective contribution of maternally-inherited mRNAs and maternally-inherited proteins to egg molecular cargo and to its developmental competence using pikeperch, an ecologically and commercially relevant freshwater fish species, as a model.ResultsOur data shed new light on the importance of maternally-inherited mRNAs in nervous system development suggesting that neurogenesis is a major mRNA-dependent non-genetic inheritance factor. In contrast, our results highlight a specific role of maternally-inherited proteins in immune response in ovulated eggs suggesting that maternal proteins would rather contribute to developmental success through protection of the embryo against pathogens. Further analysis revealed susceptibility of the transcriptome to modifications during the post-vitellogenic processes (i.e., final oocyte maturation and ovulation), whereas proteomic cargo remains unaffected. This may negatively affect developmental competence of the egg and possibly influence further nervous system development of the embryo.ConclusionsOur study provides novel insights into the understanding of type-specific roles of maternally-inherited molecules in fish. Here we show, for the first time, that transcripts and proteins have distinct, yet complementary, functions in the egg of teleost fish. Maternally-inherited mRNAs would shape embryo neurodevelopment and possibly the future behavior of the fish, while maternally-inherited proteins would rather be responsible for protecting the embryo against pathogens. Additionally, we observed that processes directly preceding ovulation may considerably affect the reproductive success by modifying expression level of genes crucial for proper embryonic development, being novel fish egg quality markers (e.g., smarca4 or h3f3a). These results are of major importance for understanding the influence of external factors on reproductive fitness in both captive and wild-type fish species.

As a result of evolution, various finfish species have developed different breeding strategies. However, there are some similarities, and one of them is the positive effect of ovarian fluid on spermatozoa. The opposite of this phenomenon was found in the common barbel (Barbus barbus). The present study analyzed the effect of ovarian fluid (OF), distilled water (DW) and Woynarovich solution (WS) on the motility, longevity and kinetics of barbel spermatozoa. These spermatozoa parameters were also evaluated with various dilutions of ovarian fluid (OF) in relation to distilled water [0:4 (Group OF 0%), 1:3 (Group OF 25%), 1:1 (Group OF 50%), 3:1 (Group OF 75%), 4:0 (Group OF 100%)] and spermatozoa reactivation after a 30 s (Group OFR30s 100%) treatment in ovarian fluid. The motility analysis was carried out using computer-assisted semen analysis (CASA). The negative interaction of ovarian fluid with spermatozoa motility in the same fish species was recorded for the first time. In pure ovarian fluid, the average spermatozoa motility (MOT) decreased significantly (1.40 ± 0.94%). The negative effect of ovarian fluid-to-spermatozoa motility was reversible, and after a 30 s treatment in ovarian fluid and later dilution with water, spermatozoa motility was reactivated (from 2.25 ± 0.53% vs 69.78 ± 6.02%). The use of Woynarovich solution as an activator of spermatozoa movement had a positive effect (P

This study investigated how the inhibition of certain enzymes present in ide sperm influences sperm motility and the percentage of fertilised eggs. The enzymes studied were acid phosphatase (AcP), β-N-acetylglucosaminidase (β-NAGase) and lactate dehydrogenase (LDH). None of the inhibitors affected ide sperm motility parameters. The addition of gossypol (a LDH inhibitor) caused a considerable increase in the percentage of fertilised eggs (92–95% compared to 63% in the control). The inhibition of AcP caused a considerable decrease in fertility rate – at the highest inhibitor dose, the percentage of fertilised eggs decreased to 26%. A similar effect was seen after the addition of acetamide (a β-NAGase inhibitor), but in this case the highest dose caused complete inhibition of fertilisation. The results presented here indicate the importance of AcP and β-NAGase in the process of ide fertilisation.

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.

The aim of this study was to test if a cryopreservation procedure using methanol as an internal cryoprotectant and trehalose as an external cryoprotectant could be applied to rainbow trout sperm (Oncorhynchus mykiss). Additionally, the effects of potassium ions and Tris-HCl on post-thaw sperm motility, viability, and fertilizing capacity were analyzed. In this study, we demonstrate for the first time that rainbow trout sperm can be cryopreserved using a trehalose and methanol based extender. Trehalose was found to be effective in rainbow trout sperm cryopreservation at concentrations ranging between 175 and 200 mM when supplemented with 10% methanol (v/v). The most effective dilution ratio was 1:11.5, which resulted in a final sperm concentration ranging from 0.8 to 1.6 × 109 spermatozoa mL− 1. A 15-min equilibration period of sperm in the extenders did not influence sperm motility parameters. The highest post-thaw sperm motility was found using extenders containing 175 mM trehalose (77.4 ± 10.8) and 175 mM trehalose with 40 mM KCl (79.3 ± 9.2). The supplementation of the extender with Tris-HCl and the adjustment of the pH to 7.5 and 8.5 resulted in a decrease in post-thaw motility. The post-thaw sperm motility in samples cryopreserved in an extender containing 40 mM of KCl did not decrease 60 min after thawing. The fertilizing capacity of sperm cryopreserved in 175 mM trehalose and 40 mM KCl was the highest and did not differ from fresh sperm at a sperm-to-egg ratio of 200,000:1. In conclusion, our results indicate that rainbow trout sperm can be successfully cryopreserved with the application of a trehalose based extender. The supplementation of the cryopreservation extender with 40 mM KCl resulted in prolonged post-thaw storage of the sperm, allowing prolonged use of the sperm over time for common fishery practices. Statement of relevance This work is related to fish production and conservation.

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Northern pike (Esox lucius) sperm equilibration and cryopreservation were conducted using a simple cryopreservation extender (CE) containing 185 mM trehalose, 40 mM KCl, 20 mM Tris at pH 8.5 and supplemented with 10% of methanol. Sperm was diluted tenfold (1:9; sperm:CE), and a subset of the samples was equilibrated (over 15 min) and stored on crushed ice (+4 °C). The remainder of the samples were drawn into French straws of 250 μl capacity and subsequently frozen. Thawing of sperm was carried out in a hot water bath at 40 °C for five seconds. The control group was fresh sperm that was neither diluted in CE nor frozen. After equilibration and cryopreservation, sperm motility parameters were analyzed using a Computer-Assisted Sperm Analysis system. Fertilization using different sperm:egg ratios (i.e. 400, 200, 100, 50, 25 and 10 thousand sperm per egg) was performed to determine the fertilization capacity of sperm. Sperm motility parameters of northern pike remain at the same level in fresh, equilibrated and cryopreserved samples. On the other hand, average sperm longevity was longest in equilibrated (118.0 s.) and cryopreserved (124.5 s.) sperm in comparison to fresh sperm (84.7 s.). Sperm samples equilibrated in CE achieved higher (n = 4. P

Collection of fish milt by stripping risks contamination with urine, which may negatively influence milt characteristics and quality. Urine contamination of milt may be avoided by using a catheter for sperm collection. The objective of this study was to compare basic pikeperch milt parameters, i.e., sperm concentration (SC), pH, osmolality of seminal plasma (OSM), and motility parameters of spermatozoa analyzed with a CASA system (Computer Assisted Sperm Analysis), collected either with a syringe (as the standard method of sperm collection) or with a catheter. Every parameter of pikeperch milt collected with a catheter was significantly higher (P

In a natural environment, seminal plasma provides spermatozoa with protection against reactive oxygen species. Storing semen in cooling conditions requires diluting it with various buffer solutions. Therefore, the protective role of seminal plasma is not sufficient enough. Semen obtained from five male specimens was diluted with the Kobayashi buffer solution at a 1:9 ratio. To determine the influence of antioxidants on semen storage, a buffer solution was used, as before, with the addition of 1 % albumin, 1 mM vitamin C, 1.5 mg ml−1 vitamin E, 5 mM sodium citrate, 5 mM glutathione and 5 mM cysteine. After the preparation of such tests, the parameters of spermatozoa motility were measured every 3–5 days, using the CASA system (Image House CRISMAS Company Ltd.). Among all used antioxidants, the best effects were observed after the addition of glutathione to semen. After 17 days of storage, the percentage of motile spermatozoa in the samples preserved with glutathione addition was 57 %, while without antioxidant addition, it was 44 %. Furthermore, the addition of cysteine and albumin also resulted in the lengthening of the life span of perch sperm cells. The presence of the remaining antioxidants (vitamins C and E, and sodium citrate) did not have any positive influence on spermatozoa viability, and in these samples, no motile spermatozoa were observed after 12 days of storage. Our data show that dilution of perch sperm with buffered solution might be a promising method for short-term storage.

Summary The aim of this study was to isolate and characterise the enzyme s-N-acetylglucosaminidase (β-NAGase) in rainbow trout and Siberian sturgeon milt, in order to find similarities or differences in its involvement in the fertilization process of these two species. While sturgeon sperm possess an acrosome, teleosts do not. Applying ion exchanging chromatography to β-NAGase of rainbow trout sperm, two peaks of β-NAGase activity were obtained from the plasma material and from the sperm. The molecular weight was 74 kDa for both milt plasma forms and 127 kDa for rainbow trout spermatozoa peak. Optimum pH of purified rainbow trout enzyme peaks ranged from 4.6 to 5.0. Michaelis–Menten constants were 11.59 × 10−4 and 11.48 × 10−4 m for I and II milt plasma peaks. Incubation of purified peaks I and II at 56°C resulted in a 45 to 50% decrease in activity of both forms. Km value for rainbow trout spermatozoa β-NAGase was 5.4 × 10−4 m. Enzyme incubation tests at high temperature proved this form of the enzyme to be most resistant to high temperatures, since its activity after 20 min of incubation at 56°C decreased only by 25%. The molecular weight of the enzyme originating from Siberian sturgeon milt plasma was about 265 and 113 kDa for I and II milt plasma peaks, respectively, and 271 kDa for the sperm extract. Optimum pH for all sturgeon purified peaks ranged from 3.8 to 5.0. The Km constant was 5.12 × 10−4, 5.28 × 10−4 and 5.0 × 10−4 m for I and II milt plasma and spermatozoa peaks, respectively. The loss of s-NAGase activity at 56°C was 58% for both milt plasma peaks and 35% for the sturgeon spermatozoa peak. The kinetic parameters, especially thermostability of s-NAGase was similar to the homologous enzymes observed in mammalian semen.

This paper presents the first data on ammonia excretion by fed and starved juvenile common tench. The aim of the study was to determine the amount of ammonia excretion by juvenile common tench Tinca tinca (L.), fed with a commercial feed under intensive rearing conditions. Rearing was conducted for a period of 4 weeks at a water temperature of 27 °C. On the test day, the amounts of ammonia excreted by starved fish (over 12 h: control group) and fish fed with a morning feed dose (3 % of biomass: experimental group) were determined at 1, 3, 5, 7, 9 and 11 h after feeding. Ammonia excretion measurements were taken in 7-day intervals. Despite the increased growth rate in successive weeks of experiment, the amount of ammonia excreted by the fish was always at a similar level within the control (maximum 0.26 mg g−1) and the experimental group (maximum 0.39 mg g−1). This paper presents for the first time reliable amount of ammonia excreted by fed and starved juvenile common tench. The obtained results may also increase the effectiveness of intensive rearing procedures (taking into account stocking density and feeding regime) and allow to design the most effective biofiltration capacity of recirculating aquaculture systems for the commercial production of this species.

Summary The study purpose was the analysis of barbel Barbus barbus (L.) milt quality and quantity with regard to the time following stimulation with [(D-Ala6, Pro9NEt)-mGnRH+metoclopramide] i.e. Ovopel. Selected parameters such as total volume of milt (TVM, ml), volume of milt per kg of body weight (VOM, ml kg−1 b.w.), sperm concentration (×109 ml−1), total sperm production (TSP, ×109), osmolality (mOsm kg−1) and pH of seminal plasma were determined. Sperm motility was analyzed by the CASA system, i.e. the percentage of sperm motility (MOT, %) and their progressive motility (PRG, %), curvilinear velocity (VCL, μm s−1) and straight line velocity (VSL, μm s−1), amplitude of lateral head displacement (ALH, μm), and beat cross frequency (BCF, Hz). Milt was collected from 12 specimens (N = 12), with the first portion obtained 12 h following treatment with Ovopel (1 granule kg−1 b. w.). Subsequent portions of milt were taken at 24 h intervals, i.e. after 36, 60, 84, 108, and 132 h. The control group (Control, N = 12) was injected with 0.9% NaCl at 0.5 ml kg−1b.w. from which milt was taken 12 h post injection. The highest TVM, VOM and TSP values were recorded 12 h after Ovopel treatment (3.2 ± 0.7 ml, 36.7 ± 10.5 ml kg−1 b.w. and 39.1 ± 9.4 × 109, respectively); lowest values were recorded after 132 h (0.8 ± 0.4 ml, 11.1 ± 6.5 ml kg−1b.w. and 13.7 ± 7.5 × 109, respectively). The highest seminal plasma osmolality values (300.0 ± 42.6 mOsm kg−1) as well as the lowest sperm concentration (12.5 ± 1.5 × 109 ml−1) were observed 12 h after Ovopel treatment. No significant differences in the percentage of sperm motility (MOT) were noted during any of the periods after hormonal stimulation, however, a change in the character of their movement (PRG) was observed. The lack of significant differences (P > 0.05) in VCL and VSL values between 12 h and 60–132 h indicates that the lengthening of time does not lead to a decrease in sperm velocity and, therefore, is not likely to have a negative impact on their quality. The highest ALH (1.9 ± 0.2 μm) and BCF (11.5 ± 1.1 Hz) values were observed when the effect of stimulation was most noticeable, i.e. 12 h after Ovopel treatment. Based on the total milt volume and sperm production, the best time for milt collection from barbel is 12 h post-hormonal treatment; 84 h post-hormonal treatment, TVM, VOM, TSP and some CASA parameters decreased, which suggest the same aging process in sperm.

The aim of this work was to compare the effects of controlled reproduction of cultured and wild common barbel, Barbus barbus (L.). Preparations containing different GnRH analogues and dopamine receptor antagonists (Ovopel, Ovaprim) as well as human chorionic gonadotropin (hCG) (in the case of cultured fish) were applied and their influence on ovulation, spermiation and quality of gametes obtained was determined. No differences in the qualitative or quantitative parameters of semen were found between fish stimulated with different hormonal preparations and those not receiving hormonal stimulation. The high suitability of Ovaprim for ovulation induction in (cultured and wild) barbel was confirmed. The highest synchronisation of ovulation was obtained after the application of Ovopel (18 ± 3 h), but the best results of controlled reproduction (expressed as the percentage of ovulations and survival of embryos) were obtained by applying Ovaprim (83.2 ± 4.1). A significantly higher percentage of ovulation was obtained in cultured fish (80–90%) than in wild fish (< 25%).