Multi-genome comparisons reveal gain-and-loss evolution of anti-Mullerian hormone receptor type 2 as a candidate master sex-determining gene in Percidae.

BACKGROUND: The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii, and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. RESULTS: We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex-determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplicates (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been likely lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome 18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variations (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex-determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in the testis than in the ovary. CONCLUSIONS: Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Neurodevelopment vs. the immune system: Complementary contributions of maternally-inherited gene transcripts and proteins to successful embryonic development in fish.

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.

Domestication affected stress and immune response markers in Perca fluviatilis in the early larval stage.

It is already known that domestication modifies stress and immune responses in juveniles and adults of several fish species. However, there is a lack of information on whether these modulations result from adaptability along the life cycle or if they are pre-determined in very early developmental stages. To shed light on mechanisms that help to explain the process of domestication, a study was conducted to analyze comparatively Eurasian perch larval performance, stress, and immune status between wild and domesticated specimens. Eurasian perch larvae obtained from wild and domesticated (generation F5 reared in recirculating aquaculture systems) spawners were reared in the same conditions during the main rearing trial (MRT) and also subjected to a thermal challenge (TC). During the study, larval performance (including survival, growth performance, swim bladder inflation effectiveness, deformity rate), the expression of genes involved in immune and stress response, and the specific activity of oxidative stress enzymes (during MRT only) were analyzed. No significant differences in hatching rate, deformity rate, or swim bladder inflation effectiveness between wild and domesticated larvae were found, whereas specific growth rate, final total length, and wet body weight were significantly lower in wild larvae. Higher mortality was also observed in wild larvae during both MRT and TC. The data obtained in this study clearly indicated that during domestication, significant modifications in stress and immune response, such as complement component c3, were noted as early as just after hatching. Generally, domesticated fish were characterized by a lower stress response and improved immune response in comparison to the wild fish. This probably resulted from the domesticated larvae being better adapted to the conditions of artificial aquaculture. The data obtained provided information on how domestication affects fish in aquaculture, and they contribute to the development of efficient selective breeding programs of Eurasian perch and other freshwater teleosts.

Domestication modulates the expression of genes involved in neurogenesis in high-quality eggs of Sander lucioperca.

Pikeperch, Sander lucioperca, is a species of high interest to the aquaculture. The expansion of its production can only be achieved by furthering domestication level. However, the mechanisms driving the domestication process in finfishes are poorly understood. Transcriptome profiling of eggs was found to be a useful tool allowing understanding of the domestication process in teleosts. In this study, using next-generation sequencing, the first pikeperch transcriptome has been generated as well as pikeperch-specific microarray comprising 35,343 unique probes. Next, we performed transcriptome profiling of eggs obtained from wild and domesticated populations. We found 710 differentially expressed genes that were linked mostly to nervous system development. These results provide new insights into processes that are directly involved in the domestication of finfishes. It can be suggested that all the identified processes were predetermined by the maternally derived set of genes contained in the unfertilized eggs. This allows us to suggest that fish behavior, along with many other processes, can be predetermined at the cellular level and may have significant implications on the adaptation of cultured fish to the natural environment. This also allows to suggest that fish behavior should be considered as a very important pikeperch aquaculture selection trait.

Repeated hormonal induction of spermiation affects the stress but not the immune response in pikeperch (Sander lucioperca).

Hormonal induction of spermiation, previously reported to be immunogenic in fishes, is a common hatchery practice in pikeperch, Sander lucioperca. The aim of the present study was to investigate the effects of repeated induction of spermiation in pikeperch, following application of either human chorionic gonadotropin (hCG) or salmon gonadoliberine analogue (sGnRHa) on sperm quality indices as well as on immune and stress response. Mature males of pikeperch (n = 7 per group) were stimulated twice with five days between injections of either hCG (hCG; 500 IU kg-1), sGnRHa (sGnRHa; 50 µg kg-1) or NaCl (control group; 1 ml kg-1) to assess spermatozoa motility with a computer-assisted sperm analysis (CASA) system. During second sampling, blood plasma was sampled for humoral innate immune (peroxidase and lysozyme activities, ACH50), stress (cortisol, glucose) and endocrine (testosterone) markers. In addition, the head kidney was dissected to assay the expression of several immune genes (such as il1, c3, hamp, tnf-α and lys genes). The results indicate that hormonal treatment significantly increased sperm production. Sperm sampled after the hormonal treatment maintained its quality throughout the study, regardless of the sampling time. However, it appears that the application of hCG induced elevated cortisol and glucose plasma levels compared to the control group. Almost all immune markers, except the relative expression of hepcidin (hamp gene), were unaffected by the two hormones applied. The results showed that the induction treatment of spermiation processes in pikeperch resulted in an important physiological stress response for which the intensity varied according to the hormonal agent used. However, this stress response (more profound following application of hCG) was weakly associated with innate immune functions. On the other hand, a significant negative correlation between the expression of several important immune markers (peroxidase activity, relative expression of c3 and il1 genes) and sperm quality indices indicates significant involvement of immune status on sperm quality. The results obtained shed light on immune-system-induced modifications to sperm quality. The data presented here highlight the need for careful revision of broodstock management and selection practices where welfare status as well as individual predispositions of fish to cope with the stress should be taken under the consideration.

The type of spawning agent affects the egg composition during out-of-season spawning but not during in-season spawning in Eurasian perch, Perca fluviatilis.

The aim of the study was to verify the effect of various hormonal agents [human chorionic gonadotropin (hCG) and salmon gonadoliberine analogue (sGnRHa)] applied at different stages of maturity of the females [out-of-season (maturation stage I) and in-season spawning (maturation stages II and IV)] on the proximate composition (PC) and fatty acid (FA) profile of eggs of Eurasian perch, Perca fluviatilis. The egg samples (7 samples from each group) were also collected from spontaneous spawning (without hormonal treatment) fish representing each maturation stage (I, II and IV for groups C-I, C-II and C-IV, respectively). The results were also compared with the eggs collected in nature (seven randomly chosen egg samples from natural spawning; group NS). Embryonic survival rate was recorded and analysis of PC and FA profile were performed, for all the groups. Embryonic survival rate varied among the groups, and only differences (P<0.05) between group C-I and NS were recorded. In-season spawning operation did not affect PC and FA profiles. Application of hCG or spontaneous spawning (group C-I) were found to have the highest effect on the FA profile. It concerned mostly total n-3 polyunsaturated fatty acids, but also stearic (C18:0), oleic [C18:1(cis9)], linoleic [C18:2(n-6)], arachidonic [C20:4(n-6)] and docosahexaenoic[C22:6(n-3)] acids. The application of sGnRHa during out-of-season spawning had the lowest effect on the FA profile. The results presented indicate that controlled reproduction can affect the FA profile only during out-of-season spawning. This negative effect can be presumably compensated by the application of sGnRHa as a spawning agent.

Paternal identity impacts embryonic development for two species of freshwater fish.

Paternal, compared to maternal, contributions were believed to have only a limited influence on embryonic development and larval fitness traits in fishes. Therefore, the perspective of male influence on early life history traits has come under scrutiny. This study was conducted to determine parental effects on the rate of eyed embryos of Ide Leuciscus idus and Northern pike Esox lucius. Five sires and five dams from each species were crossed using a quantitative genetic breeding design and the resulting 25 sib groups of each species were reared to the embryonic eyed stage. We then partition variation in embryonic phenotypic performance to maternal, paternal, and parental interactions using the Restricted Maximum Likelihood (REML) model. Results showed that paternal, maternal, and the paternal×maternal interaction terms were highly significant for both species; clearly demonstrating that certain family combinations were more compatible than others. Paternal effects explained 20.24% of the total variance, which was 2-fold higher than the maternal effects (10.73%) in Ide, while paternal effects explained 18.9% of the total variance, which was 15-fold higher than the maternal effects (1.3%) in Northern pike. Together, these results indicate that male effects are of major importance during embryonic development for these species. Furthermore, this study demonstrates that genetic compatibility between sires and dams plays an important role and needs to be taken into consideration for reproduction of these and likely other economically important fish species.

Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla).

Vitrification was successfully applied to the sperm of two fish species, the freshwater Eurasian perch (Perca fluviatilis) and marine European eel (Anguilla anguilla). Sperm was collected, diluted in species-specific non-activating media and cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). Additional sperm quality parameters such as sperm head morphometry parameters (in case of European eel) and fertilizing capacity (in case of Eurasian perch) were carried out to test the effectiveness of vitrification. The vitrification method for Eurasian perch sperm resulting the highest post-thaw motility (14±1.6%) was as follows: 1:5 dilution ratio, Tanaka extender, 30% cryoprotectant (15% methanol+15% propylene-glycol), cooling device: Cryotop, 2µl droplets, and for European eel sperm: dilution ratio 1:1, with 40% cryoprotectant (20% MeOH and 20% PG), and 10% FBS, cooling device: Cryotop, with 2µl of sperm suspension. Viable embryos were produced by fertilization with vitrified Eurasian perch sperm (neurulation: 2.54±1.67%). According to the ASMA analysis, no significant decrease in head area and perimeter of vitrified European eel spermatozoa were found when compared to fresh spermatozoa.

Improvement of common carp (Cyprinus carpio) sperm cryopreservation using a programable freezer.

The applicability of a programmable freezer for the increased-scale cryopreservation of common carp sperm was investigated. The effect of different equilibration times, cryopreservation methods, extenders, dilution ratios, activating solutions on the post-thaw motility of common carp sperm was investigated. The suitable post-thaw storage time-interval as well as fertilizing capacity of cryopreserved sperm was also examined. The motility, curvilinear velocity (VCL) and straightness (STR) values did not decrease significantly during 60min of equilibration neither in equilibrated nor thawed groups. Motility parameters of thawed sperm were similar using a conventional cryopreservation technique using a polystyrene box [motility (33%), VCL (47µm/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54µm/s) and STR (89%)]. The highest motility and VCL was measured with a sugar based extender (grayling extender) at a ratio 1:9 (motility: 52%, VCL: 76µm/s) and 1:20 (motility: 49%, VCL: 76µm/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2min. A storage time of six hours following thawing did not have a significant effect on the motility parameters of thawed carp sperm. Agglutination was observed during cryopreservation of an elevated volume of sperm whereas motility 47%, VCL 62µm/s and STR 91% were measured after thawing. Fertilization rate with thawed sperm (32%) was significantly lower compared to the control group (73%). According to our results, the developed method using a programmable freezer is suitable for the cryopreservation of elevated number of straws. However, carp sperm agglutination during freezing may have a negative effect on the fertilizing capacity.

Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish.

In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability.

Paternal-effect-genes revealed through sperm cryopreservation in Perca fluviatilis.

Knowledge about paternal-effect-genes (PEGs) (genes whose expression in the progeny is influenced by paternal factors present in the sperm) in fish is very limited. To explore this issue, we used milt cryopreservation as a specific challenge test for sperm cells, thus enabling selection amidst cryo-sensitivity. We created two groups of Eurasian perch (Perca fluviatilis) as a model - eggs fertilized either with fresh (Fresh group) or cryopreserved (Cryo group) milt from the same male followed by phenotypic-transcriptomic examination of consequences of cryopreservation in obtained progeny (at larval stages). Most of the phenotypical observations were similar in both groups, except the final weight which was higher in the Cryo group. Milt cryopreservation appeared to act as a "positive selection" factor, upregulating most PEGs in the Cryo group. Transcriptomic profile of freshly hatched larvae sourced genes involved in the development of visual perception and we identified them as PEGs. Consequently, larvae from the Cryo group exhibited enhanced eyesight, potentially contributing to more efficient foraging and weight gain compared to the Fresh group. This study unveils, for the first time, the significant influence of the paternal genome on the development of the visual system in fish, highlighting pde6g, opn1lw1, and rbp4l as novel PEGs.

Ovarian alterations in wild northern pike Esox lucius females.

The aim of the present study was to analyse the occurrence of macroscopically visible ovary alterations in 2 populations of northern pike Esox lucius L. originating from lakes in the Mazurian Lake District (NE Poland). The alterations were characterised by ovary tissue that was morphologically malformed, in part or in whole, and contained immature oocytes, i.e. trophoplastic or previtellogenic oocytes instead of vitellogenic oocytes. These alterations were found only in the ovaries, and no morphological alterations of the testes were noted. Macroscopic and histological analyses were carried out in order to classify the observed alterations in the ovaries. Three types of alterations were identified in which morphological malformations as well as histological investigation of the ovaries were considered. An analysis of the size and age of the fish in relation to the occurrence of alterations as well as of the macroscopic and histological nature of the alteration types was made. The data obtained revealed no lake or age dependency of the observed alterations. Based on the results obtained, we suggest that the presence of endocrine disruptors in the environment or/and genetic factors could be responsible for these kinds of gonad anomalies. However, our results did not allow us to determine the aetiology of the alterations.

The effectiveness of Ovaprim, Ovopel, and their combinations in artificial reproduction of common rudd Scardinius erythrophthalmus under controlled conditions.

In this study, a practical protocol for artificial reproduction was developed for the rudd, Scardinius erythrophthalmus, to help to produce this fish for aquaculture industries and also for restocking programs. For this purpose, the efficiency of different hormonal agents and their combinations for spawning induction of female breeders of rudd was evaluated. Ovulations were stimulated using different treatments as: two injections of Ovaprim (Ova), two injections of Ovopel (Ovo), a priming dose of Ovopel with a resolving dose of Ovaprim (Comb1), a priming dose of Ovaprim with a resolving dose of Ovopel (Comb2), and two injections of sterile 0.9% NaCl solution as a control group. During the study, ovulation success, egg developmental competence as well as the performance of the freshly hatched larvae were recorded. There was no ovulation in females from the control group. In hormone-treated groups, 84-100% of fish ovulated, but no differences were observed in ovulation success (P > 0.05). The shortest latency period in treated fish was recorded in the Ovo group (431.6 degree-hour, P < 0.05). The best results in terms of working fecundity (number of obtained eggs per fish), relative fecundity, fertilization success, hatching rate, and embryo survival up to the eyed stage were achieved in the Ova and Comb1 groups (P < 0.05). Moreover, better survival rates up to the absorbing yolk sac stage and the lowest abnormality of larvae were observed in Ova and Comb1 groups (P < 0.05). There was no significant difference in the incubation time among different groups (P > 0.05). The obtained results indicate that two injections of Ovaprim and/or a priming dose of Ovopel with a resolving dose of Ovaprim are suitable for the artificial reproduction of rudd female breeders. Due to the proper effects and ease of use, two injections of Ovaprim could be recommended for the production of this species for restocking or aquaculture programs.

Squalene Supplementation as a Novel to Increase PUFA Content in Fish Tissues.

Squalene is an antioxidant that plays an essential role in fat metabolism. The study aimed to assess the effect of squalene supplied in feed on the growth performance, health status, and fatty acid profiles of muscle and liver of Siberian sturgeon, rainbow trout, and Eurasian perch. The experimental feeds containing 0%, 0.5%, and 1.0% squalene were prepared for each fish species. Hematological and biochemical indices, liver histology, and fatty acid profiling of muscle and liver were analyzed. Squalene supplementation was safe for fish, and no negative influence on growth status was observed. However, changes in the values of hematological and biochemical indicators were found, including the level of triglycerides in the blood of rainbow trout, and cholesterol in the blood of Eurasian perch. The addition of squalene influences the nucleocytoplasmic index values in all fish offered feed containing 1% squalene. The retention of squalene in the liver and muscle of experimental Siberian sturgeon was observed in both 0.5% and 1.0% squalene levels of feed. The PUFA and docosahexaenoic acid increase was observed in all fish in groups with squalene addition. Dietary squalene increases the content of PUFAs in tissues of the examined species.

Multi-genome comparisons reveal gain-and-loss evolution of the anti-Mullerian hormone receptor type 2 gene, an old master sex determining gene, in Percidae.

The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis, Perca schrenkii and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene (amhr2bY), previously suggested to be the master sex determining (MSD) gene in P. flavescens. Phylogenetically related and structurally similar amhr2 duplications (amhr2b) were found in P. schrenkii and Sander lucioperca, potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus, this amhr2b duplicate has been lost while it was subject to amplification in S. lucioperca. Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens. In P. fluviatilis, a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome-18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variants (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three (c18h1orf198, hsdl1, tbc1d32) with higher expression in testis than ovary. Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Cortical reaction as an egg quality indicator in artificial reproduction of pikeperch, Sander lucioperca.

The aim of this study was to investigate the process of the cortical reaction in eggs of pikeperch, Sander lucioperca (L.), as well as the application of microscopic assessment of this process in egg quality evaluation. The analysis was carried out with eggs obtained from 10 females by artificial reproduction, in which hormonal stimulation with hCG was applied. Subsequently, each sample of eggs (separately from each female fish) was analysed. The analysis included observation of the cortical reaction and the process of egg swelling, and determination of the effect of temperature (12, 14 and 16°C) and the presence of spermatozoa on the cortical reaction. The results indicate that the cortical reaction in pikeperch eggs is quite violent, resulting in visible deformation of eggs between 3 and 5 min after activation. No effect of temperature or the presence of spermatozoa on the cortical reaction was observed. A strong correlation was recorded for the percentage of egg deformations observed and embryo survival rate. The described method of determination of pikeperch egg quality (based on egg deformation rate between 3 and 5 min after activation) may be highly useful, both in scientific research (where high-quality eggs are required) and in hatchery practice.

Artificial reproduction of Caspian roach, Rutilus caspicus following stimulating ovulation with Ovaprim, Ovopel, and their combinations under controlled conditions.

The aim of this study was to evaluate the efficiency of different hormonal agents and their combinations for spawning induction of females Caspian roach, Rutilus caspicus. Five groups of fish were injected intraperitoneally as follows: two injections of Ovaprim (T1), two injections of Ovopel (T2), a priming dose of Ovopel with a resolving dose of Ovaprim (T3), a priming dose of Ovaprim with a resolving dose of Ovopel (T4), and two injections of sterile 0.9% NaCl solution as a control group. The results showed that no fish ovulated in the control group. In hormone-treated groups, no differences were observed in ovulation success (P > 0.05). The shortest latency time and highest working and relative fecundities were observed in fish stimulated with two injections of Ovaprim (T1 group). The mean values of fertilization success in treated fish were in the range of 87.9-93.1% and the highest values were observed in T3 and T1 groups (P < 0.05). The best results in terms of ovulation index (92.1%), hatching rate (93.7%), and survival up to the eyed egg stage (89.4%) were achieved after administering two injections of Ovaprim (T1 group). There were no significant differences in the incubation time and embryos survival rates up to absorbing the yolk sac among different groups (P > 0.05). Overall, the best results of controlled reproduction of Caspian roach were obtained after using two injections of Ovaprim. Consequently, the Ovaprim is more suitable for restocking and aquaculture purposes of endangered populations of Caspian roach.

The effectiveness of human chorionic gonadotropin in stimulation of second spermiation in pikeperch Sander lucioperca during the spawning season.

The aim of the present study was to evaluate the effectiveness of human chorionic gonadotropin (hCG) in stimulation of second spermiation in already reproduced pikeperch males during the same spawning season. Fish (mean weight 919.9 ±â€¯236.9 g) were divided into the control saline-injected group, and two groups injected with 200 and 400 IU hCG/kg body weight. Forty-eight hours following injection (at a temperature of 16.7 °C) the percentage of fish producing useable amounts of semen increased from 30% in the control group up to 80% in the treated fish and the highest volume of semen was recorded in fish treated with 400 IU/kg BW hCG. Spermatocrit (50.7 ±â€¯9.1%) and sperm concentration (17.5 ±â€¯5.0 × 109 spermatozoa/mL) were significantly higher in control fish than those recorded in hCG-treated fish (24.8 ±â€¯7.7% and 8.1 ±â€¯2.4 × 109 spermatozoa/mL in 200 IU/kg; 32.3 ±â€¯17.6% and 10.9 ±â€¯7.5 × 109 spermatozoa/mL in 400 IU/kg). No significant differences were observed between the groups with respect to sperm motility (as a spermatozoa activity time and percentage); however, a change in plasma alkaline phosphatase activity was found in the group injected with 400 IU/kg of hCG. The lack of significant differences in plasma metabolites and cortisol between control and hCG-treated fish indicated that the hCG had no effect on the stress response. Testosterone levels were significantly increased in the hCG-treated fish, whereas no significant differences in 17ß-estradiol were recorded. No differences between the groups in plasma levels of thyroid hormones suggested lack of hCG-induced effect on hypothalamus-pituitary-thyroid axis. The results of this study demonstrated that the spermiation of already reproduced pikeperch males could be induced by hCG during the same spawning season. Based on the results on semen volume and concentration, injection with hCG could improve the efficiency of pikeperch reproduction to reduce the number of brooders and costs of juvenile pikeperch production.

Fish with larger pre-seasonal oocytes yields lower egg quality in season - A case study of outdoor-cultured domesticated Pikeperch (Sander lucioperca).

Seasonal reproduction of domesticated pikeperch has been the most critical spawning batch in several European countries. The present study aimed to monitor oocyte growth between mid-November and seasonal spawning to evaluate if oocyte growth trends may predict egg quality. Nineteen sexually mature females were monitored for oocyte sizes every two months. In mid-March, fish were transported to the indoor facility and artificially reproduced. Sixteen females ovulated and egg quality parameters were assessed and further related to oocyte sizes measured in November, January, and March, as well as to size increments between samplings. Based on the oocyte diameters, fish were assigned to two size groups, and the egg quality was compared. Oocyte growth was greater between the first two samplings compared to the consecutive period (150.1 ± 16.5 µm vs 24.7 ± 20.4 µm). Diameters assessed in November and March positively correlated with oil globule fragmentation, while diameters assessed in November and January positively correlated with larval malformation. Although in January larger oocytes showed better embryo survival than smaller ones (60.6 ± 9.5% vs 37.8 ± 23.2%,) they were characterized with greater percent of malformed larvae (25.0 ± 22.0% vs 5.4 ± 3.9%). It appears that fish with bigger oocytes in pre-season have an affinity toward increased embryo survival, however, they seem to have an issue with oil globule fragmentation and larvae malformation in case of seasonal spawning. Therefore, the selection of breeders for either pre-seasonal or seasonal reproduction batch according to the November oocyte size is recommended.

Optimalisation of the Activation Medium and Effect of Inhibiting Activities of Acid Phosphatase, Lactate Dehydrogenase and ß-N-Acetylglucosaminidase on the Fertilisation Success of Eurasian Perch (Perca fluviatilis L.).

Although methods for the artificial reproduction of perch have been developed, a lack of information remains regarding the enzymes present in its semen, as well as their role in the fertilisation process. In this study, we first select the optimal activating solution for perch fertilisation and then determine the inhibition effect of enzymes that have already been reported as present in the sperm of teleosts-acid phosphatase (AcP), lactic dehydrogenase (LDH) and ß-N-acetylglucosaminidase (ß-NAGase)-on the percentage of motile spermatozoa and fertilised eggs. Of the 8 studied activation media, a solution composed of 80 mM NaCl, 20 mM KCl, 10 mM Tris, with pH 8.0 and 206 mOsm/kg proved to be optimal for perch gametes. The addition of ammonium molybdate (AcP inhibitor) caused no significant reduction in the percentage of fertilised eggs. On the other hand, the addition of 0.25 mM gossypol (LDH inhibitor) and 0.125 M acetamide (ß-N-acetylglucosaminidase inhibitor) significantly decreased the fertilisation percentage to 41.1% and 52.4%, respectively, in contrast to the control (89.9 %). Both LDH and ß-NAGase thus seem to play a very important role in the perch fertilisation process.