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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 µg/kg) and resolving (25 µg/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 17α,20β-dihydroxy-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.

Domestication is a condition in which the breeding, care and feeding of animals are, at least in part, controlled by humans. Information regarding the changes in the protein composition of eggs in response to domestication is very limited. Such data are prerequisite for improvements in the reproduction of domesticated fish. The aim of this study was to examine the impact of domestication on the proteome of pikeperch eggs using two-dimensional differential in-gel electrophoresis. We analysed high-quality eggs from domesticated and wild pikeperch fish to reveal proteins that were presumably only related to the domestication process and not to the quality of eggs. Here, we show that domestication has a profound impact on the protein profile of pikeperch eggs. We identified 66 differentially abundant protein spots, including 27 spots that were more abundant in wild-caught pikeperch eggs and 39 spots that were enriched in eggs collected from domesticated females. Eggs originating from wild-caught females showed higher expression levels of proteins involved in folding, apoptotic process, purine metabolism and immune response, whereas eggs of domesticated females showed higher expression levels of proteins that participated mainly in metabolism. The changes in metabolic proteins in eggs from domesticated females can reflect the adaptation of pikeperch to commercial diets, which have profoundly distinct compositions compared with natural diets. The decrease in the abundance of proteins related to immune response in eggs from the domesticated population suggests that domestication may lead to disturbances in defence mechanisms. In turn, the lower abundance of heat shock proteins in eggs of domesticated fish may indicate their adaptation to stable farming conditions and reduced environmental stressors or their better tolerance of stress from breeding. The proteins identified in this study can increase our knowledge concerning the mechanism of the pikeperch domestication process.

Control of the reproduction of domesticated stocks is considered a prerequisite for aquaculture development of pikeperch. However, knowledge about the physiology of the captive pikeperch male reproductive system and the biology of semen is very limited, especially regarding protein characteristics. The aims of our study were to characterize pikeperch sperm quantity and quality parameters and to analyze changes in the proteome of the same males spawned for the first and second times. Moreover, attempts were made to generate the first proteomic library of seminal plasma proteins. Semen collected during the first spawning season was characterized by lower sperm concentration and volume than for the second season. Using mass spectrometry-based label-free quantitative proteomics, we identified 850 proteins in the seminal plasma of pikeperch from both spawning seasons, and 65 seminal proteins were found to be differentially abundant between the first and second spawning seasons. The majority of differentially abundant proteins were involved in stress and immune responses, developmental processes, cofactor metabolic processes, proteolysis, cellular oxidant detoxification and organization of the extracellular matrix (ECM). In addition, several proteins unique to pikeperch seminal plasma were identified, including antifreeze proteins, hibernation-specific plasma proteins, lectins and vitellogenin. In summary, our results indicate that males that spawned for the first time were characterized by incompletely mature gonads and the expression of proteins associated with the early phase of spermatogenesis and ECM organization. On the other hand, males that spawned for the second time exhibited advanced gonadal maturation and expression of proteins related to the late stage of spermatogenesis and sperm maturation, including regulation of reactive oxygen species generation, bicarbonate production, sperm elongation and separation. The identification of a large number of seminal plasma proteins provides a valuable resource for understanding the functions of seminal plasma and the molecular mechanisms involved in testicular development and maturation in domesticated fish, which is a prerequisite for better control of reproduction in captivity.

Pikeperch (Sander lucioperca) is a highly valuable fish in Europe. However, development of aquaculture of pikeperch is highly limited due to seasonality of production. This can be overcome by the controlled reproduction of domesticated fish. The first steps of domestication process may induce changes at anatomical, physiological and molecular levels, thereby affecting a variety of biological functions. While there is abundant literature on their effects on stress and growth for example, these effects on reproduction received limited attention notably in pikeperch, a promising candidate for the development of aquaculture. To answer the question of this life-history effect on pikeperch’s reproduction, we compared two groups (weight: 1 kg) originated from Czech Republic and with the same domestication level (F0). The first group was a recirculating aquatic system cultured one (2 years, previously fed with artificial diet, never exposed to natural changes in temperature/photoperiod conditions) and the second one was a pond cultured group (3 to 4 years, bred under natural feeding and temperature/photoperiod). The wild group successfully spawned, while the farmed one did not spawn at all. During the program, gonadosomatic indexes of both males and females were significantly higher for the wild fish, as well as the sexual steroids. Gene expression analysis revealed significantly lower LH transcript levels at the pituitary level for the farmed females and lower FSH transcript levels at the pituitary level for the males. In conclusion this study showed that the previous rearing conditions (e.g. culture system, age, diet, etc.) alter the further progress of gametogenesis and the reproductive performances in response to controlled photothermal program for both sexes in pikeperch.

In pikeperch, Sander lucioperca, aquaculture hormonal treatment is usually applied to synchronize ovulation. However, the effect of dopamine (DA) receptor antagonists, in particular those blocking the D1 DA receptors, remains unknown. Thus, the aim of the present study was to investigate and compare the effects of D1 and D2 DA receptor antagonists on the sex-steroid production and reproductive performance of the species. Two experiments were performed during which mature pikeperch females were injected with different molecules: NaCl 0.9% (negative control) or human chorionic gonadotropin 500 IU/kg (positive control) in both experiments, metoclopramide (a D2 receptor antagonist; 4 mg/kg or 20 mg/kg) or SCH23390 (a D1 receptor antagonist; 0.8 mg/kg or 4 mg/kg) alone (experiment 1) or in combination with a salmon gonadotropin-releasing hormone analogue (sGnRHa at 25 µg/kg; experiment 2). In experiment 2, fish were also injected with sGnRHa (25 µg/kg) as positive control. Samplings of oocytes and blood were performed on the day of injection and after 24 h (both experiments), after 48 h (experiment 2) and at the time of ovulation (both experiments). In non-ovulating fish, samplings were performed 7 days (experiment 1) or 14 days (experiment 2) after injection. In experiment 2, various zootechnical parameters of fertilized eggs were recorded (survival, hatching and malformation rates). The two antagonists alone were ineffective in inducing the final stages and regulating sex-steroid (testosterone, 11 ketotestosterone, 17β estradiol and 17,20β-dihydroxy-4-pregnen-3-one) production. When administered with sGnRHa, both SCH23390 and metoclopramide induced the final stages. However, only SCH23390 stimulated testosterone (4 mg/kg) and 17β estradiol (0.8 mg/kg) production compared with sGnRHa alone. None of the treatments affected the survival, hatching or malformation rates. This is the first report suggesting that in pikeperch the D1, but not the D2, DA receptor antagonist would be involved in the testosterone and 17β estradiol production as a potentiator of the sGnRHa effect.

Ova ageing is the most important factor affecting fish egg quality after ovulation. Long-term storage of fish ova, using cryopreservation and vitrification techniques, has been unsuccessful to date. Instead, short-term in vitro ova storage has been used successfully and optimized in some cultured fish species. In vitro ova storage can drastically improve mass production of larvae and juveniles in the hatcheries by providing the possibility of the synchronous artificial fertilization for different females. To study how long unfertilized eggs of Eurasian perch (Perca fluviatilis L.) can retain their fertilizing ability after stripping, eggs were stored at temperatures of 4°C, 8°C and 12°C for 72 h post-stripping (HPS). The stored eggs of four female perch were separately fertilized at 0 h (i.e. control eggs fertilized before storage) and at 6-hour intervals during the experimental period of 72 h. The embryos reaching the eyed-egg and hatched-larvae stages, eyed-egg mortality and larval malformation rates were recorded as indices of egg quality. The results indicated that the maximum eyed eggs and hatched larvae (86% and 63%, respectively) were observed for eggs fertilized immediately after stripping, whereas the storage of the eggs at 4°C for 48 HPS decreased the eyed-egg and hatched-larvae rates to 46% and 17%, respectively. The use of a higher storage temperature resulted in a more rapid decrease in egg viability: eyed-egg and hatched-larvae rates of 23% and 9%, respectively, were obtained after 48 HPS storage at 8°C and 2% and 1% for eggs stored at 12°C. Eyed-egg mortality and larval malformation rates were not significantly affected by post-stripping ova ageing for at least up to 36 h. Thereafter, both values increased significantly and were measured to be the highest in the most aged ova. The present study demonstrated that stripped Eurasian perch eggs can be stored for at least 12 h at 4°C to 12°C without a significant reduction in their quality.

Aquaculture in rural areas, carried out in accordance with current EU requirements, aims to contribute significantly to the conservation of the biodiversity of aquatic resources, the protection of which is a prerequisite for sustainable economic and social development. The objective of this study was to present the conceptual and technical framework and to analyze the costs and profitability of producing the consumer-attractive Eurasian perch ( Perca fluviatilis L.) based on the untapped potential of hatchery infrastructure and dedicated earthen ponds for common carp ( Cyprinus carpio L.) production. The experiments were conducted under Polish conditions, but the results/methods can be successfully applied in other countries, mainly in Central and Eastern Europe, where production in earthen ponds is the basis of aquaculture production. The analysis of the profitability of the venture shows that the cost structure was dominated by labor and feed inputs. Despite high variable costs, this type of production can be profitable and contribute to food security and economic development in line with the EU's Blue Growth Strategy. The article also analyzes the potential opportunities and risks of fish production based on the Recirculating Aquaculture System (RAS) and the Recirculating Aquaculture Multitrophic Pond System (RAMPS).

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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 a mhr2 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., Competing Interests: COMPETING INTERESTS All authors declare no competing interests.

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.

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 ± 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 ± 9.1%) and sperm concentration (17.5 ± 5.0 × 10 9 spermatozoa/mL) were significantly higher in control fish than those recorded in hCG-treated fish (24.8 ± 7.7% and 8.1 ± 2.4 × 10 9 spermatozoa/mL in 200 IU/kg; 32.3 ± 17.6% and 10.9 ± 7.5 × 10 9 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 17β-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., Competing Interests: Conflict of interest The authors declare that there is no conflict of interests for the present study., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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 β-N-acetylglucosaminidase (β-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 (β-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 β-NAGase thus seem to play a very important role in the perch fertilisation process.

Background: Considering wild inter-populational phenotypic differentiation can facilitate domestication and subsequent production of new species. However, comparing all populations across a species range to identify those exhibiting suitable key traits for aquaculture (KTA; i.e. important for domestication and subsequent production) expressions is not feasible. Therefore, proxies highlighting inter-populational divergences in KTA are needed. The use of such proxies would allow to identify, prior to bioassays, the wild population pairs which are likely to present differentiations in KTA expressions in aquaculture conditions. Here, we assessed the relevance of three alternative proxies: (i) genetic distance, (ii) habitat divergence, and (iii) geographic/hydrologic distances. We performed this evaluation on seven allopatric populations of Perca fluviatilis for which divergences in KTA had already been shown., Results: We showed differences in the correlation degree between the alternative proxy-based and KTA-based distance matrices, with the genetic proxy being correlated to the highest number of KTA. However, no proxy was correlated to all inter-populational divergences in KTA., Conclusion: For future domestication trials, we suggest using a multi-proxy assessment along with a prioritisation strategy to identify population pairs which are of interest for further evaluation in bioassays., (© 2021. The Author(s).)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Eurasian perch (Perca fluviatilis Linnaeus, 1758) is native to almost entire Eurasia. For over the last two decades, this species became an important candidate for intensive freshwater aquaculture due to its high consumer's acceptance and overall market value. Hence, the intensive production of Eurasian perch has increased considerably allowing effective domestication; there is still a need for the development of effective selective breeding programmes allowing its further expansion. This process, in turn, can be significantly facilitated by molecular genetics. The genetic information of Eurasian perch and its populations is limited. Up to date information of regarding genetic diversity of many populations is still missing, including microsatellites for Eurasian perch, which could be useful during the selective breeding programmes allowing parental assignment and/or to follow heritability of desired traits. In this study, we have developed and characterized new polymorphic microsatellites. Subsequently, those 12 markers have been used further to compare two Hungarian and one Polish Eurasian perch populations. The Hungarian stocks had high genetic similarity (with low diversity), as we assumed, while the Polish population differed significantly. All populations deviated significantly from the Hardy-Weinberg equilibrium, and heterozygote deficiency was detected in all, showing the presence of an anthropogenic effect., (© 2021. The Author(s).)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2020 Wiley Periodicals LLC.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To date, a comparative analysis of larval performance and digestion abilities between wild and domesticated Eurasian perch has not yet been performed. Eurasian perch larvae from wild and domesticated spawners were reared in the same conditions and at different development stages, growth performance variables, the expression of genes encoding digestive enzymes and specific enzymatic activity were analysed. No significant differences in hatching rate, deformity rate or swim bladder inflation effectiveness between wild and domesticated larvae were found. Specific growth rate, final total length and wet body weight were significantly lower in wild larvae, whereas higher mortality in wild larvae was observed compared to domesticated larvae. The data obtained in this study clearly indicate that during domestication, significant modification of digestion ability occurs at the very beginning of ontogeny, where domesticated fish are characterised by lower enzymatic activity and lower expression of genes encoding digestive enzymes. This probably results from the low diversity of the food offered in culture conditions, which significantly modified digestion capability. The obtained data provide an understanding of how domestication affects fish in aquaculture and may improve the planning of selective breeding programs of Eurasian perch and other freshwater Teleosts.

The objectives of the present study were to determine values for semen quality variables in the Eurasian perch (i.e., osmolality of seminal plasma as well as sperm motility characteristics analyzed with CASA system) in response to (1) the method of milt collection (stripping or catheterization) and (2) experimental contamination of catheterized semen with urine (0%, 5%, 10%, 20%, 30% and 50% of contamination). Additionally, the effect of short-term chilled storage of experimentally contaminated semen (during the 24 h post semen collection period) on motility characteristics was investigated. Use of a typical stripping procedure resulted in about 5%-10% contamination of semen with urine, what is much less compared with other species. Markedly lesser values of straight line velocity (VSL) and consequently less linearity of spermatozoa movement (LIN) in perch semen, however, occurred as a result of stripping (46 ± 4 μm/s and 38 ± 4% for VSL and LIN, respectively), when compared to sperm collected by catheterization (87 ± 5 μm/s and 77 ± 2% for VSL and LIN, respectively), indicate that even a 10% contamination of semen with urine may have negative effects on quality. Exposure of semen to urine resulted in a significant dose-dependent decrease in the percentage of motile spermatozoa (MOT) and both velocity variables (VSL and VCL). Amount of urine contamination also affected MOT, VCL, VSL and LIN value during short-term storage. In conclusion, it is important to avoid semen contamination by urine when using the stripping procedure in the Eurasian perch, either for controlled reproduction or sperm preservation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The effect of carp pituitary homogenate (CPH, n = 7) at a dose of 2.0 mg kg -1 and (D-Arg 6 , Pro 9 NET)-sGnRH + domperidone (Ovaprim, n = 7) at a dose of 0.5 ml kg -1 in northern pike (Esox lucius) sperm maturation under controlled conditions was examined. On the control group, 0.9% NaCl at a dose of 1.0 ml kg -1 (n = 7) was used. Sperm was collected 48 h following injection. Sperm quantity (total sperm volume, total sperm production and sperm concentration), sperm motility using the Computer-assisted sperm analysis (CASA) system and sperm and seminal plasma quality (sperm pH, seminal plasma osmotic pressure and seminal plasma pH) were determined for each male separately in each group. The results of the present study demonstrate that hormonal treatment had a positive effect on sperm maturation in northern pike, regardless of the hormonal preparation used. However, even though no differences were found in total sperm volume and total sperm production between fish injected with either CPH or Ovaprim, it should be highlighted that the highest progressive motile sperm (PRG), straight-linear velocity (VSL) and movement linearity (LIN) was noted in fish treated with Ovaprim. It was also found that it is possible to collect sperm from non-hormonally manipulated fish. However, in such a case, only a small sperm volume (around 0.1 ml) characterised by lowered PRG (below 40%) was noted. Considering the fact that only after Ovaprim application sperm motility with progressive movement of sperm above 50% was observed, the hatchery practice of collection of sperm 48 h after its application (at a dose of 0.5 ml kg -1 ) may be recommended., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study analysed (i) the effect of human chorionic gonadotropin (hCG) and salmon gonadoliberine analogue (sGnRHa) on the effectiveness of induction of spermiation and (ii) the effect of latency time following the application of those spawning agents on the quantity and quality of the sperm of Eurasian perch, Perca fluviatilis, obtained during out-of-season spawning. For this study, pond-reared fish were used which had been acclimated to the controlled conditions. Three groups were distinguished which were treated with either saline (0.9% NaCl; control group), hCG (500 IU kg -1 ) or sGnRHa (100 μg kg -1 ). The fish were kept in a recirculating system at 12 °C throughout the study, during which sperm was collected every two days between the 2nd and 10th day following hormonal treatment. During the study, quantitative (e.g. sperm volume, total sperm production) and qualitative (measured with a computer-assisted sperm analysis system - i.e. CASA) parameters were monitored. The results of the study indicate that the hormonal treatment had a highly beneficial effect on the spermiation rate (100% in experimental groups from day 6 following injection) as well as quantity, which increased 50% in experimental groups (over 2200 × 10 9 of spermatozoa per kg of body weight) by day 4 following injection. For the sperm quality, both spawning agents tested had a rather positive effect, although sperm motility rate (MOT) was seen to be significantly reduced on day 10 following the application of hCG (MOT = 72.8% ± 8.1), which was not observed after the application of sGnRHa (minimum mean MOT 81.7% ± 6.1). The results clearly indicate that hormonal treatment had a positive effect on spermiation in Eurasian perch, most apparent from day 6 following injection, regardless of the hormonal agent used. Though application of sGnRHa allowed a high volume of high quality sperm to be stripped for two days longer (up to day 10 post-injection) compared to the application of hCG., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Variable and low egg quality is a major limiting factor for the development of efficient aquaculture production. This stems from limited knowledge on the mechanisms underlying egg quality in cultured fish. Molecular analyses, such as transcriptomic studies, are valuable tools to identify the most important processes modulating egg quality. However, very few studies have been devoted to this aspect so far. Within this study, the microarray-based transcriptomic analysis of eggs (of different quality) of sea bass (Dicentrarchus labrax) was performed. An Agilent oligo microarray experiment was performed on labelled mRNA extracted from 16 batches of eggs (each batch obtained from a different female) of sea bass, in which over 24,000 published probe arrays were used. We identified 39 differentially expressed genes exhibiting a differential expression between the groups of low (fertilization rate < 60 %) and high (fertilization rate > 60 %) quality. The mRNA levels of eight genes were further analyzed by quantitative PCR. Seven genes were confirmed by qPCR to be differentially expressed in eggs of low and high quality. This study confirmed the importance of some of the genes already reported to be potential molecular quality indicators (mainly rnf213 and irf7), but we also found new genes (mainly usp5, mem-prot, plec, cenpf), which had not yet been reported to be quality-dependent in fish. These results suggest the importance of genes involved in several important processes, such as protein ubiquitination, translation, DNA repair, and cell structure and architecture; these probably being the mechanisms that contribute to egg developmental competence in sea bass.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The quality and fertilizing capacity of perch (Perca fluviatilis) sperm collected outside of the spawning season (off-season) and cryopreserved at a commercial scale, were tested. Basic parameters (equilibration time, dilution ratio, sperm concentration, post-thaw motility duration) which can have a significant effect on cryopreservation success were systematically investigated for effects on sperm quality using computer assisted sperm analysis (CASA). No significant decrease in progressive motility (pMOT) and straightness (STR) of fresh-diluted sperm was recorded among groups equilibrated for 0, 30 or 60min in an extender with cryoprotectants. Curvilinear velocity (VCL) was reduced significantly after 30min (30min: 146±15μm/s, 60min: 124±18μm/s) of equilibration compared to the control (174±9μm/s). After thawing, no decrease in pMOT or VCL was observed at different equilibration times in any of the analyzed groups. No correlation was observed among progressive motility, dilution ratios (p=0.7) and cell concentrations (p=0.1). The use of different activating solutions resulted in similar pMOT and VCL in the first 120s post-thaw. Nevertheless, post-thaw sperm motility was reduced after 30s using all activators. Motility parameters with low variation were recorded after thawing of 57 straws (pMOT: 37±7%, VCL: 92±10μm/s, STR: 89±3%). Ten randomly selected straws from commercial-scale cryopreservation resulted in a high fertilization rate (cryopreserved sperm: 72±14%, fresh control: 94±2%). An optimized commercial-scale cryopreservation protocol was successfully developed for Eurasian perch. The applicability of the off-season collected perch sperm for cryopreservation and fertilization was demonstrated., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Standardization of fertilization protocols is crucial for improving reproductive techniques for externally fertilizing fish in captive breeding. Therefore, the objectives of this study were to determine the effects of preincubation of eggs and activation medium on the percentage of eyed embryos for ide (Leuciscus idus). Pooled eggs from five females were preincubated in three different activating media for 0, 30, 60, 90, and 120 seconds and then fertilized by pooled sperm from five males. At the eyed-egg stage, the percentage of viable embryos was later calculated. Results showed that preincubation time was significant for the freshwater activation medium (P < 0.001), such that the percentage of eyed embryos declined across the preincubation time gradient. Additionally, there was an effect on the percentage of eyed embryos when eggs were incubated with Woynarovich solution (P < 0.001), such that a decline was detected at 90 seconds, whereas no effect was detected for the saline water medium. Activating medium had a significant effect on the percentage of eyed embryos for each preincubation time (P < 0.05). More precisely, freshwater produced the lowest percentage of eyed embryos at all preincubation times (ranged from 1.9% at 120 seconds to 43.6% at 0 seconds), whereas saline water and Woynarovich solution produced the highest percentage of eyed embryos at 0 seconds and 30 seconds before incubation. Woynarovich solution produced the highest percentage of eyed embryos at 60 seconds (65.26%), whereas saline water produced the highest percentage at 90 seconds (68.37%). No difference was detected between saline water and Woynarovich solution at 120 seconds. Examination of sperm traits showed no impact of activating medium on computer assisted sperm analysis parameters. Together, these results suggest that saline water or Woynarovich solution improve fertilization rate in ide during IVF; thus, these media are useful for standardizing fertilization protocols and controlled reproduction for this species., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Two different cryopreservation methods were compared and an optimal dilution ratio for the use of controlled-rate freezer (CRF) was established for Eurasian perch (Perca fluviatilis) sperm. Progressive motility (72 ± 15%) and curvilinear velocity (VCL, 146 ± 11 μm/s) of sperm cryopreserved with CRF did not reduce significantly compared to fresh sperm [progressive motility (90 ± 4%), VCL (173 ± 24 μm/s)]. On the other hand, progressive motility (62 ± 15%) and VCL (120 ± 21 μm/s) of sperm cryopreserved with the conventional floating frame technique were significantly lower when compared to the fresh control. Sperm in both cryopreserved groups showed significantly higher straightness [STR, CRF (84 ± 4%), frame (84 ± 2%)] than in the fresh control group (68 ± 4%). Perch sperm cryopreserved with CRF at a dilution ratio of 1:20 showed significantly higher progressive motility (49 ± 6%) than at a ratio of 1:5 (39 ± 6%) and showed significantly higher VCL (129 ± 11 μm/s) than at dilution ratios of 1:10 (112 ± 17 μm/s) and 1:5 (115 ± 9 μm/s)., (Copyright © 2014 Elsevier Inc. All rights reserved.)