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Mitotic gynogenesis results in the production of fully homozygous individuals in a single generation. Since inbred fish were found to exhibit an increased frequency of body deformations that may affect their survival, the main focus of this research was to evaluate the ratio of individuals with spinal deformities among gynogenetic doubled haploids (DHs) brown trout as compared to nonmanipulated heterozygous individuals. Gynogenetic development was induced by the activation of brown trout eggs by UV-irradiated homologous and heterologous (rainbow trout) spermatozoa. The subsequent exposure of the activated eggs to the high hydrostatic pressure disturbed the first cleavage in gynogenetic zygotes and enabled duplication of the maternal haploid set of chromosomes. The survival rate was significantly higher among gynogenetic brown trout hatched from eggs activated with the homologous UV-irradiated spermatozoa when compared to DHs hatched from eggs activated by the heterologous spermatozoa. More than 35% of the gynogenetic larvae exhibited body deformities, mostly lordosis and scoliosis. The percentage of malformed brown trout from the control group did not exceed 15%. The increased number of deformed larvae among DHs brown trout suggested rather a genetic background of the disease related to the fish spine deformities; however, both genetic and environmental factors were discussed as a cause of such conditions in fish.

Poor quality eggs produced by the fully homozygous doubled haploids (DHs) may impair generation of clonal lines in fish species. In the present research, gynogenetic development of rainbow trout (Oncorhynchus mykiss) was induced in eggs originated from the DH females. Eggs were activated with the UV-irradiated grayling (Thymallus thymallus) spermatozoa and subjected to the high hydrostatic pressure (HHP) shock to provide diploid clonal individuals. Only two of four DH females produced eggs that were successfully activated by the irradiated spermatozoa and subsequently developed into the gynogenetic embryos. Survival rates of rainbow trout from the clonal lines equalled 21.5% and 19.8% during embryogenesis and decreased after hatching to 18.6% and 14.9%, respectively. Some of the dead rainbow trout clones collected between hatching and swim-up stage were emaciated and exhibited spinal deformities including scoliosis. Provided results confirmed limited developmental competences of eggs produced by rainbow trout DH females. Clonal rainbow trout developing in such eggs exhibited reduced survival and increased frequency of the body abnormalities.

To gain insight into the possible implications for aquaculture of the presence of ZEN in fish feed at the EC guidance level, we determined the effects of over two months of feeding of premarket size rainbow trout (~ 250 g) with ZEN-contaminated feed at a dose of 1.81 mg·kg − 1 feed (90.5% of the current guidance value) under aquaculture conditions. After 37 and 71 days of the experiment, both control and ZEN-challenged fish were weighed and measured to assess their growth performance. To examine the health-related effects of ZEN exposure, fish blood was collected for biochemical measurements of plasma markers, and their livers and gonads were fixed for histopathological analysis. Additionally, to examine the effect of ZEN on the molecular background of fish reproduction, the mRNA level of vitellogenin (vtg) in the fishes' liver was determined. Finally, exposed fish tissues were analyzed for residual concentrations of ZEN and its metabolites in order to estimate the potential health risks for the fish consumers. Our results indicate that when premarket size fish are fed for two months with ZEN-contaminated feed at a concentration just below the EC guidance value, the growth of the fish is not affected. The analysis of residual concentrations of ZEN indicated that there was no threat to the health of potential consumers of the meat from the exposed fish. However, the residuals accumulated in the fishes' intestine and liver, and trace amounts of ZEN were also found in their ovaries. Although histological analysis of the liver cross-sections revealed structural irregularities in the treated group, including necrotic areas, disorders of polygonal hepatocytes, cytoplasm vacuolization, and macrophage aggregates, these developments were not accompanied by alterations in levels of plasma biochemical markers. This shows that ZEN had only a limited influence on the liver structure of the exposed fish. Although the experimental groups did not differ in terms of vtg mRNA levels, our results suggest that ZEN in feed may accelerate sexual maturation of the female fish, as ovarian development was more advanced in the exposed group. Our findings suggest that the presence of ZEN in fish feed may have consequences for aquaculture and warrant the need for further research in order to reassess the recommended limits. Statement of relevance Current feed guideline may have consequences for aquaculture.

Meiotic gynogenetic development of the rainbow trout (Oncorhynchus mykiss, Walbaum 1792) was induced in the course of egg activation performed by the UV-irradiated homologous and heterologous (European grayling Thymallus thymallus Linnaeus, 1758) spermatozoa. To recover diploidy in the gynogenetic zygotes, activated eggs were subjected to the high pressure shock in order to inhibit extrusion of the second polar body. Gynogenetic rainbow trout progeny hatched from the eggs activated by the irradiated rainbow trout and grayling milt with similar hatching rates of 28.19% and 29.22%, respectively. However, gynogenetic rainbow trout produced with grayling semen had shown lower survival than gynogenotes provided with the homologous spermatozoa during two years of rearing. Viable hybrids are not produced between rainbow trout and grayling which ensured that fish obtained in this experiment were true gynogenetic progenies. A Robertsonian polymorphism characteristic for the rainbow trout from the studied strain was also observed among the gynogenetic specimens that exhibited diploid chromosome number ranging from 58 to 62 and stable chromosome arm number (FN= 104). No radiation-induced fragments of the paternal chromosomes were observed in the gynogenetic individuals. Fish produced in both experimental variants were genotypic (XX) and phenotypic (gonads) females. The results confirmed that the gynogenetic protocol used in the present research is an efficient means of producing all-female gynogenetic rainbow trout stocks.

Contents Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation-induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 ± 2.4% and 16.14 ± 2.90% and the survival rates at the first feeding stage of 10.48 ± 3.48% and 12.78 ± 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiation-induced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes.

Rainbow trout (Oncorhynchus mykiss Walbaum) and sea trout (Salmo trutta Linnaeus, 1758) show large karyotypic differences and their hybrid offspring is not viable due to unstable karyotype and chromosome fragmentation. However, gametes from these two species were used to induce gynogenetic development. Rainbow trout eggs activated by UV-irradiated sea trout sperm were subjected to high hydrostatic pressure (HHP) shock to prevent release of the 2nd polar body (early shock) or to inhibit the first cleavage (late shock) in order to produce diploid meiotic gynogenotes and gynogenetic doubled haploids (DHs), respectively. Cytogenetic analysis proved fish that development was induced by the sea trout spermatozoa were rainbow trout. In turn, molecular examination confirmed homozygosity of the gynogenetic DHs. Presumed appearance of the recessive alleles resulted in lower survival of the gynogenetic DH larvae (~25%) when compared to survival of the heterozygous (meiotic) gynogenotes (c. 50%). Our results proved that genomic incompatibilities between studied trout species result in the hybrid unviability. However, artificial gynogenesis including activation of rainbow trout eggs with UV-irradiated sea trout spermatozoa was successfully induced. As both species are unable to cross, application of the UV-irradiated sea trout spermatozoa to activate rainbow trout development assures only maternal inheritance with no contamination by the residues of the paternal chromosomes.

To investigate whether ZEN transfers from the alimentary tract of fish to the somatic cells of ovaries or the oocytes, mature females of rainbow trout were orally exposed to ZEN at a dose of 1 mg·kg − 1 body mass. At sampling times of 2, 6, 12, 24, 48, and 96 h, tissues of the fish (intestine, liver, ovaries, oocytes, muscles, and plasma) were extracted to determine the concentration of ZEN and its metabolites using immunoaffinity columns and HPLC-FLD. Our results confirm that ZEN is transferred from the alimentary tract to the reproductive system of the fish, and indicate that the mycotoxin concentrates in the somatic cells of the ovaries. Importantly, ZEN transferred to the fishes' oocytes and muscles only to a limited extent. Our additional survey of fish hatcheries and local stores indicated only trace amounts of ZEN residuals in the samples that were collected in Poland and Norway between 2013 and 2015, which probably reflects good hygienic conditions for the feed used in these hatcheries. Furthermore, our results indicate that the health risk from dietary intake of ZEN from fish roe is negligible. However, the potential of ZEN to transfer to the fish ovaries may be of concern for aquaculture.

We tested the usefulness of 0.18 M glucose in 9% methanol as an extender for the cryopreservation of semen of sex-reversed rainbow trout females (n = 9). The semen-to-extender ratio of 1:9 and 15 min equilibration were used. Motility and viability parameters as well as the fertilization rate at the eyed stage and hatching were assessed for post-thaw sperm. The fertilization success of cryopreserved semen was about 80% for both 500,000 and 1,000,000:1 sperm-to-egg ratios. The application of the cryopreservation procedure resulted in remarkably high post-thaw sperm motility and viability (55 and 56%, respectively). Our results for the first time show that the post-thaw fertilization ability of rainbow trout sex-reversed female semen can be similar to that of fresh semen at a sperm-to-egg ratio as low as 500,000:1. The sperm motility pattern characterized by low linearity, high amplitude of lateral head displacement and curvilinear velocity seems to be beneficial for the fertilizing ability of thawed sperm. Sperm viability appears to be a reliable indicator of the quality of thawed semen of rainbow trout sex-reversed females. The cryopreservation procedure using glucose–methanol extender after scaling up can be recommended for routine hatchery practice.

The goal of this study was to improve post-thaw quality of cryopreserved rainbow trout semen. Quality was assessed by measuring sperm motility and fertilization rates at the eyed, hatching, and swim-up larvae stages. We first tested how varying glucose concentrations in a methanol extender (0.1, 0.2, and 0.3 M, n = 9 different males per concentration) and different semen-extender ratios (1:3, 1:1, and 3:1, n = 3 males and in a separate experiment 1:3 and 1:5, n = 6) affected post-thaw motility. Sperm motility and fertilization rates of samples with 0.18 M glucose in 9% methanol as an extender, a 1:5 sperm-to-extender ratio, and a 15 min equilibration period then were measured at sperm-to-egg ratios of 100,000, 300,000, and 600,000:1. The optimal cryopreservation conditions were found to be 0.15 M glucose in the extender, a 15 min equilibration period, and a 1:5 semen-to-extender ratio. This combination resulted in high post-thaw motility (49.9%) and hatching rates (67.1 ± 18.7, 78.6 ± 17.0, and 84.4 ± 10.0% for sperm-to-egg ratios of 100,000, 300,000 and 600,000:1, respectively). To our knowledge, this is the first report to demonstrate that the post-thaw fertilization ability of rainbow trout semen can be similar to that of fresh semen at a sperm-to-egg ratio as low as 600,000:1.

Potassium ions are known to have an inhibitory effect on the sperm motility of salmonids. For this reason, the addition of K(+) to the extender is frequently applied. However, the effect of the addition of K(+) to the extender has not yet been tested. The aim of this study was to test the influence of potassium ion supplementation of the extender on the sperm motility parameters from five Salmonidae species (rainbow trout (Oncorhynchus mykiss), sex-reversed female rainbow trout, whitefish (Coregonus lavaretus), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis)). Semen samples were diluted in extender containing 0.18 M glucose in 9% methanol (GM) supplemented with 0, 20 or 40 mM potassium chloride. After thawing sperm were stored for 30, 60, 120 and 240 min at 4 °C. Our results demonstrated that the presence of potassium ions in the extender had a negative effect on percentage of motile sperm in four of the salmonid species. In contrast, potassium ions appeared to have a positive effect on percentage of post-thaw motile sperm in whitefish semen. However, this effect could be mimicked by changing the osmolality of the extender (which was achieved by increasing the glucose concentration to 0.22 M). The addition of potassium ions turned out to have no positive effect on post-thaw storage time. Our results suggest that osmolality, rather than potassium ions, seems to be essential for cryopreservation success of salmonids sperm. Further studies should focus on the effects of small changes in osmolality on the post-thaw quality of semen.