Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814).

Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3-6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.

Induction of gynogenetic and androgenetic haploid and doubled haploid development in the brown trout (Salmo trutta Linnaeus 1758).

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.

Use of eggs derived from the interspecific charr hybrids to induce androgenetic development of the brook charr (Salvelinus fontinalis Mitchill 1814).

Although, brook charr (Salvelinus fontinalis Mitchill 1814) and Arctic charr (Salvelinus alpinus Linnaeus 1758) are able to cross and give fertile offspring, their androgenetic nucleocytoplasmic hybrids are not viable. To overcome incompatibility between the egg cytoplasm of one charr species and the sperm nucleus of another charr species, application of F1 interspecific hybrids as egg donors for the purpose of androgenesis has been proposed. Here, androgenetic development of the brook charr was successfully induced in the brook charr eggs and the eggs derived from the reciprocal brook charr × Arctic charr F1 hybrids. A working androgenesis protocol included inactivation of the maternal nuclear DNA achieved by irradiation of the eggs with 420 Gy of X-rays, insemination of such treated eggs with the haploid sperm cells and exposition of the haploid androgenetic zygotes to the high hydrostatic pressure shock (51.711 MPa for 4 min) applied 420 min after insemination. Androgenetic larvae that hatched from the brook charr and the hybrid eggs were shown to be homozygous brook charr individuals. Androgenetic individuals exhibited 84 chromosomes and 100 chromosome arms (FN), values characteristic for the brook charr diploid cells. Strategy hybridize first than induce androgenesis should be tested in order to provide androgenetic offspring in other salmonids that are able to cross and produce fertile offspring.

Distribution of telomeric DNA sequences on the X-radiation-induced chromosome fragments observed in the genome of androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814).

Cytogenetic screening of the androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814) offspring hatched from eggs exposed to 420 Gy of X-radiation before insemination exhibited residues of the irradiated maternal nuclear genome in the form of small chromosome fragments. Remnants of the irradiated chromosomes had different sizes, and their number varied intraindividually from 1 to 15. To efficiently pass through the series of the cell divisions, such chromosome fragments must have had functional kinetochores. Distribution patterns of the telomeric hybridization signals on the chromosome fragments enabled us to distinguish their 3 groups: (i) telomere-less ring chromosomes with fused broken chromosome arms, (ii) rings formed in the course of fusion of the radiation-broken chromosome arm with the opposite telomeric region and exhibiting interstitial telomeric signals at the fusion point, and (iii) chromosome fragments with fused unprotected sister chromatids of 1 broken arm and intact telomeres from the other arm. Disturbances during segregation of such fragments, mainly breakages during anaphase, may partially explain intraindividual variation in the number and size of the chromosome fragments observed in the androgenetic brook trout.

Influence of zearalenone on selected biochemical parameters in juvenile rainbow trout (Oncorhynchus mykiss).

Zearalenone (ZEA) is a mycoestrogen frequently found in food and animal feed materials all over the world. Despite its hydrophobic character, ZEA is also found in surface and ground waters which suggests an environmental risk for aquatic animals. Knowledge concerning mycotoxin-related mechanisms of toxicity is still incomplete, e.g. little is known about the influence of ZEA exposure on fish. The aim of this study was to investigate the effect of ZEA on selected biochemical parameters in juvenile rainbow trout after 24, 72, and 168 h of intraperitoneal exposure (10 mg/kg of body weight). The analysis showed a slight tendency towards prolonged blood clotting time and significant iron deficiency in the liver and ovary of exposed animals. However, no differences in aminotransferase (AlaAT, AspAT) activity or glucose levels in fish plasma was observed. The results of this study suggest that although trout exposed to ZEA did not exhibit any distinct symptoms of liver damage, the mycotoxin tested was able interfere with blood coagulation and iron-storage processes.

Ovarian transcriptome analysis of diploid and triploid rainbow trout revealed new pathways related to gonadal development and fertility.

Triploidisation represents several advantages (e.g. sterility) and therefore is routinely applied in aquaculture of several commercially important fish species, including rainbow trout. The comparative transcriptomic analysis of ovaries of triploid (3N) and diploid (2N) female rainbow trout revealed a total of 9 075 differentially expressed genes (DEGs; 4 105 genes upregulated in 2N and 4 970 genes upregulated in 3N ovaries, respectively). Identified clusters for DEGs upregulated in 3N and 2N ovaries were different, including carbohydrate and lipid metabolic process and transport, protein modification, signalling (related to folliculogenesis) and response to stimulus for DEGs upregulated in 2N, and developmental process, signalling (related to apoptosis, cellular senescence and adherence junctions) and regulation of RNA metabolic process for DEGs upregulated in 3N. The enrichment of processes involved in carbohydrate and lipid metabolism in 2N ovaries indicated high metabolism of ovarian tissue and the energy reservoir generation indispensable during the earliest stages of development. Our results highlight the importance of oocyte hydration along with oestrogen, insulin, leptin, fibroblast growth factor, and Notch signalling and pathways related to the regulation of cyclic adenosine monophosphate (cAMP) levels in proper oocyte meiotic maturation prior to ovulation in 2N ovaries. Conversely, triploidisation may lead to an increase in ovarian cellular senescence and apoptosis, which in turn can result in abnormal gonadal morphology and fibrosis. The downregulation of genes responsible for the precise regulation of meiosis and proper chromosome segregation during meiosis probably affects meiotic maturation via irregular meiotic division of chromosomes. The induction of triploidy of the rainbow trout genome resulted in enhanced expression of male-specific genes, genes responsible for re-establishing the transcriptional balance after genome reorganisation and genes involved in regulatory mechanisms, including gene silencing and DNA methylation. To the best of our knowledge, this is the first genome-wide investigation providing in-depth comprehensive and comparative gene expression patterns in the ovary from 2N and 3N rainbow trout females helping in elucidating the molecular mechanisms leading to impaired gonadal development and sterility of female triploids.

Formation of chromosome aberrations in androgenetic rainbow trout Oncorhynchus mykiss.

Residues of maternal nuclear DNA in the form of chromosome fragments were observed in the healthy and morphologically normal androgenetic rainbow trout Oncorhynchus mykiss. A hypothetical model for formation of chromosome re-arrangements caused by the incomplete maternal nuclear DNA inactivation in the androgenetic rainbow trout was proposed in the present paper.

Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa.

Preservation of DNA integrity is essential for protection of sperm quality. This study examined, with the use of comet assay, DNA fragmentation of rainbow trout (Oncorhynchus mykiss) spermatozoa subjected to UV irradiation (2,075 microW/cm(2), 0-15 min) or oxidative stress induced by hydrogen peroxide (0-20mM). Sperm motility and fertilizing ability were also measured. A dramatic increase in DNA fragmentation was recorded after 5 min UV irradiation but no significant changes in sperm motility were observed at this time. Longer irradiation resulted in a decrease in motility parameters and further increase of DNA fragmentation. UV irradiation caused a clear decrease in the percentage of eyed embryos and most of the embryos did not hatch. When highly diluted sperm suspensions (50,000-fold) were exposed to 0.1mM H(2)O(2) evident increase in DNA fragmentation was observed. On the other hand, when more concentrated sperm suspensions (diluted only 40-fold) were employed (in order to conduct motility and fertilization measurements at the same time) 1-20mM H(2)O(2) caused only moderate increase in DNA fragmentation and dose-dependent decline in sperm motility and fertilizing ability. This suggests that toxic effects of H(2)O(2) were primarily related to inhibition of sperm motility. Our results demonstrate that comet assay can be used for monitoring the effectiveness of fish sperm DNA inactivation by UV irradiation. Therefore, the comet assay together with sperm motility analysis can be applied in optimization works of gynogenetic procedures in fish. Lack of effectiveness of H(2)O(2) in inducing major DNA fragmentation suggests presence of mechanisms of antioxidative defense in rainbow trout spermatozoa.

Effect of postthaw storage time and sperm-to-egg ratio on fertility of cryopreserved brook trout sperm.

The aim of this study was to test the influence of postthaw storage time on sperm motility parameters of brook trout (n = 9). Furthermore, we examined the effect of sperm-to-egg ratios of 300,000:1 and 600,000:1 on fertility of postthaw, cryopreserved, brook trout sperm. The application of a cryopreservation procedure produced very high postthaw sperm motility (56.8 ± 4.0%). The cryopreserved sperm of brook trout could be stored up to 60 minutes without loss of the percentage of sperm motility (52.0 ± 9.0%). The fertilization capacity of brook trout postthaw sperm was comparable with the fertilization rate of fresh semen at a sperm-to-egg ratio as low as 300,000:1 (42.4 ± 14.0% and 36.5 ± 11.0% for eyed and hatched stages, respectively). The possibility of postthaw semen storage for the prolonged time and the obtainment of high fertilization rate at low sperm-to-egg ratio can lead to the significant improvement of brook trout semen cryopreservation procedure.

Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm.

Milt of brown, rainbow and brook trout was cryopreserved. Activity of aspartate aminotransferase (AspAT) and acid phosphatase was assayed both in supernatants and in spermatozoa obtained from thawed sperm samples; additionally, post-thaw motility was evaluated. Enzyme activities differed according to fish species and were strongly affected by the type of cryoprotectant used. The activity in supernatants was usually higher than that in spermatozoa because of protein leakage from injured cells. AspAT activity in cryopreserved spermatozoa correlated positively with fertilization success in all three species. There was a negative correlation between activity of extracellular (supernatant) AspAT and fertilization rates in variants with dimethyl sulfoxide and dimethylacetamide-based extenders. The motility of thawed sperm, determined microscopically, provided some information on the cryopreservation efficiency of trout milt.

Space- and time-resolved density measurements of a high-intensity laser-produced plasma for x-ray laser studies.

We present a detailed study on the spatiotemporal density evolution of a plasma created by optical-field ionization of a high-pressure pulsed gas jet by a 10-TW, 60-fs Ti:sapphire laser. The plasma dynamics has been studied on a 17-ns time scale with a 60-fs time resolution and a 5-microm space resolution using a Mach-Zehnder interferometer. The density profile and the plasma radial expansion were accurately measured for conditions relevant to x-ray laser schemes in H-like nitrogen which were recently proposed [S. Hulin et al., Phys. Rev. E 61, 5693 (2000)]. The results were reproduced well by hydrocode simulations that allowed to infer the plasma temperature.

Hot-electron influence on L-shell spectra of multicharged Kr ions generated in clusters irradiated by femtosecond laser pulses.

Strong L-shell x-ray emission has been obtained from Kr clusters formed in gas jets and irradiated by 60-500-fs laser pulses. Spectral lines from the F-, Ne- Na-, and Mg-like charge states of Kr have been identified from highly resolved x-ray spectra. Spectral line intensities are used in conjunction with a detailed time-dependent collisional-radiative model to diagnose the electron distribution functions of plasmas formed in various gas jet nozzles with various laser pulse durations. It is shown that L-shell spectra formed by relatively long nanosecond-laser pulses can be well described by a steady-state model without hot electrons when opacity effects are included. In contrast, adequate modeling of L-shell spectra from highly transient and inhomogeneous femtosecond-laser plasmas requires including the influence of hot electrons. It is shown that femtosecond-laser interaction with gas jets from conical nozzles produces plasmas with higher ionization balances than plasmas formed by gas jets from Laval nozzles, in agreement with previous work for femtosecond laser interaction with Ar clusters.

Spatially resolved x-ray spectroscopy investigation of femtosecond laser irradiated Ar clusters.

High temperature plasmas have been created by irradiating Ar clusters with high intensity 60-fs laser pulses. Detailed spectroscopic analysis of spatially resolved, high resolution x-ray data near the He(alpha) line of Ar is consistent with a two-temperature collisional-radiative model incorporating the effects of highly energetic electrons. The results of the spectral analysis are compared with a theoretical hydrodynamic model of cluster production, as well as interferometric data. The plasma parameters are notably uniform over one Rayleigh length (600 microm).

Blood cells in rainbow trout Oncorhynchus mykiss milt: relation to milt collection method and sampling period.

The presence of blood cells in milt of rainbow trout (Oncorhynchus mykiss) collected every week between the middle at the end of the spawning season, either by stripping or by catheterization was investigated. Basic sperm biological and biochemical characteristics were also evaluated. Because milt often becomes contaminated with blood during collection, we also studied the influence of experimental blood contamination on sperm motility and biochemical parameters of seminal plasma. We demonstrated the presence of blood cells (erythrocytes, lymphoid, and phagocytes) in rainbow trout milt collected by both methods. Both sampling period and collection method influenced sperm characteristics, however the relationship between these characteristics and blood cells are not clear at present. A high number of blood cells in milt was found in some samples, possibly due to inflammation, because at the same time we observed bacteria and elevated levels of protein and antiproteinase activity in contaminated samples. Experimental contamination of milt with blood did not influence sperm motility, protein concentration and LDH activity of the 5-day-stored semen. Our study demonstrated that blood cells were present in rainbow trout milt. Blood cells may also appear in milt as a result of bleeding and their elevated levels are present during inflammation.

Androgenesis in rainbow trout using cryopreserved spermatozoa: the effect of processing and biological factors.

In addition to producing homozygous lines for biomedical and genomic research and monosex stocks for commercial purposes, androgenesis is the biotechnology considered most promising and reliable for recovering complete nuclear genome information from cryopreserved fish cells. That is because procedures of cryopreserving spermatozoa, contrary to procedures for oocytes or entire eggs, are being well developed. Application of androgenesis in genome banking programs addresses the needs of both the aquaculture industry (safeguard for valuable strains and lines) and natural resource conservation (in vitro protection of endangered species or populations). The present study was focused on successful production of an androgenetic rainbow trout stock using cryopreserved spermatozoa. Our report constitutes the first time a full factorial analysis of processing and biological factors affecting androgenesis efficacy has been presented. Also for the first time, the survival of androgenetic individuals during 2 years of life was recorded. Remarkably high survival rates were observed in one of the two experiments-up to 42.5 +/- 2.8% of hatched larvae, 22.5 +/- 0.1% of swim-up larvae and 10.5% of androgenetic alevins 0.4 g. Mortality rates in androgenetic groups were high especially during the first 6 months. In all, 114 androgenetic individuals (0.9%) survived 2 years. Cryopreservation of spermatozoa generally did not affect androgenesis efficiency significantly, however, this effect was significantly dependent on the method of ploidization shock and on the duration of treatment. Significant interactions were revealed between the irradiation dose and the magnitude of pressure applied, and between the treatment of sperm and duration of pressure shock. Individual variability of spermatozoa donors significantly affected androgenesis efficiency regardless of their genetic (outbred or inbred) origin. Genetic source of the oocytes, contrary to spermatozoa, proved to be an important factor. Following findings of other researchers that androgenesis using cryopreserved spermatozoa is possible, we demonstrated that viable stock could be successfully established from cryopreserved nuclear genome information. Complex statistical analysis of previously developed procedures resulted in information-rich data regarding factorial interactions helpful for developing protocols in genome-restoration programs.

Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa.

The effects of extender composition and equilibration time on fertilizing ability of cryopreserved spermatozoa from rainbow trout, Oncorhynchus mykiss, were investigated. In addition, enzyme activity in supernatants from thawed sperm was assessed. The use of the two extenders: Erdahl & Graham's + 10% DMA (dimethyl acetamide) + 10% egg yolk and 0.3 M glucose + 10% DMA yielded the highest post-thaw fertilization rates. We observed interactions between extender constituents and the equilibration of diluted semen. This indicates a multifactorial effect of the extender constituents on spermatozoal resistance against injuries. The 10-min equilibration of spermatozoa in extender before freezing generally lowered the fertilization ability of spermatozoa, except for DMA-based extenders. The addition of egg yolk to the extender was generally beneficial, especially in DMA- and DMSO-based extenders. The use of low-density lipoprotein fraction showed no advantage to full-yolk or free-of-yolk extenders. Aspartate aminotransferase and lactate dehydrogenase leakage from damaged spermatozoa correlated negatively with the ability of cryopreserved spermatozoa to fertilize eggs. Each factor tested, when analyzed separately, did not give general information about its effect on the fertilization ability of cryopreserved sperm. The multifactorial analysis of the important factors in cryopreservation of trout spermatozoa showed their cumulative effect. This is the most likely reason for divergent information reported elsewhere on the effect of various factors in the cryopreservation of rainbow trout spermatozoa.

Cell-mediated immunity in yellow forms of rainbow trout.

The aim of the present study was to investigate possible pleiotropic effects of the genotypes controlling the palomino and albino coloration on blood phagocyte and lymphocyte activity. The results showed that the wild coloured trout has a higher metabolic and potential killing activity of blood phagocytes, compared to albino and palomino coloured trout. The proliferative response of blood lymphocytes stimulated by ConA or LPS indicated a similar pattern. The results showed that lymphocyte proliferation was statistically significantly higher in wild coloured trout, compared to albino and palomino trout.

[Hepatitis C--epidemiology, diagnosis and treatment in children]. / Wirusowe zapalenie watroby typu C--problemy epidemiologii, diagnostyki i leczenia dzieci.

OBJECTIVE: Epidemiology dates of hepatitis C in children are not well known because of usually asymptomatic course of disease. Vertical transmission has became one of the most common route of hepatitis C virus infection in children. METHODS AND MAIN OBSERVATIONS: Twenty one infants of twenty HCV positive mothers were studied from 1998 to 2000 in the Clinic of Infectious Disease in Childhood of Medical University in Warsaw. Five of the infants were HCV RNA positive. All uninfected children became HCV-antybody negative by 12 months. RESULTS AND CONCLUSIONS: Chronic infection is common in most cases of HCV infection, but the disease progression is slower than in adults. Experience of treatment of chronic hepatitis C in children is limited, with about 40% having sustained response to the interferon therapy. Combinated therapy with interferon and ribavirin may give a better response.

[Vertical HIV transmission in Poland from 1989-2000]. / Odmatczyne zakazenia HIV w Polsce w latach 1989-2000.

OBJECTIVE: To evaluate vertical HIV transmission rate in Poland. METHODS: 132 children born to HIV positive mothers were evaluated and their HIV infection status was established. MAIN OBSERVATIONS AND RESULTS: There are 60 HIV-infected children in Poland. 92% of them have been infected perinataly. From 1989 to 1994 the rate of vertical transmission was 25%. Since September 1994, when prophylactic strategies were started, the rate has decreased to 21.4% (in children with prophylaxis to 1.8%). CONCLUSIONS: Vertical HIV infection prophylactic programs rely on women's knowledge about their HIV infection status and are the only way to diminish pediatric HIV infection worldwide. The authors show difficulty of providing proper medical care of HIV positive women in Poland and underly the need of voluntary HIV testing for all women before pregnancy.

Inhibition of ß-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii).

ß-N-Acetylglucosaminidase (ß-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that ß-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of ß-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for ß-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of ß-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of ß-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that ß-NAGase can play a significant role in the fertilization process in teleosteans.