Konrad Ocalewicz

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.

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.

Activation of the Albino Sterlet Acipenser ruthenus Eggs by UV-Irradiated Bester Hybrid Spermatozoa to Provide Gynogenetic Progeny.

Meiotic gynogenesis was induced in the albino form of sterlet Acipenser ruthenus by activation of eggs with UV-irradiated bester (Huso huso x Acipenser ruthenus) spermatozoa followed by inhibition of the second meiotic division performed by a heat shock. Obtained putative gynogenetic progeny were all albinos. The genetic verification based on three microsatellite DNA markers confirmed the only maternal inheritance of the progeny from the gynogenetic experimental groups. Cytogenetic analysis proved the gynogenetic sterlets were diploids. Application of the albino phenotype together with the molecular and the cytogenetic diagnostic approaches enabled to evaluate the efficiency of the spermatozoa irradiation and application of the heat shock to restore diploid state in the gynogenetic zygotes.

High Rate of Deformed Larvae among Gynogenetic Brown Trout (Salmo trutta m. fario) Doubled Haploids

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.

Application of The UV-Irradiated Homologous and Heterologous Sperm for Activa Tion of the Rainbow Trout (Oncorhynchus Mykiss) Eggs and Production of the Gynogenetic Stocks

Abstract 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.

Genome incompatibility between rainbow trout ( Oncorhynchus mykiss ) and sea trout ( Salmo trutta ) and induction of the interspecies gynogenesis

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.

Disturbances in the ploidy level in the gynogenetic sterlet Acipenser ruthenus

Artificial mitotic gynogenesis, a chromosome set manipulation, is applied to provide the homozygous progeny with only maternal inheritance. Here, gynogenetic development was induced in the sterlet Acipenser ruthenus L. (Acipenseridae) by activation of the eggs originating from albino females with the UV-irradiated spermatozoa from wild-coloured males, followed by the heat shock applied to suppress the first mitotic division in the haploid zygotes. All experimentally obtained gynogenetic offspring possessed recessive albino coloration. Moreover, the genetic verification, based on three microsatellite DNA markers, confirmed the only maternal inheritance in the albino progeny. Cytogenetic screening enabled identification of the aneuploids, haploids, diploids, triploids, tetraploids and mosaic individuals among the gynogenetic larvae that hatched from the eggs subjected to the heat shock. Furthermore, 40% of the larvae from the haploid variants of the research that were not exposed to the temperature shock showed the diploid chromosome number. A variation of the ploidy level observed in the gynogenetic sterlets may be the consequence of the spontaneous polyploidisation that occurred in the haploid zygotes. Moreover, observation during embryogenesis showed varied stages of eggs development and the asynchronous cell cleavages that may have resulted in the chromosomal disturbances observed in the gynogenetic sterlets here.

Androgenetic development of X- and Y-chromosome bearing haploid rainbow trout embryos.

Haploid fish embryos are important in studies regarding role of the recessive traits during early ontogeny. In fish species with the male heterogamety, androgenetic haploid embryos might be also useful tool in studies concerning role of the sex chromosomes during an embryonic development. Morphologically differentiated X and Y chromosomes have been found in a limited number of fish species including rainbow trout (Oncorhynchus mykiss Walbaum 1792). To evaluate role of the sex chromosomes during rainbow trout embryonic development, survival of the androgenetic haploids in the presence of X or Y sex chromosomes has been examined. Androgenetic haploid rainbow trout were produced by fertilization of X-irradiated eggs with spermatozoa derived from the normal males (XY) and neomales, that is, sex-reversed females (XX) to produce X- and Y-bearing haploids, and all X-bearing haploids, respectively. Survival rates of the androgenetic progenies of normal males and neomales examined during embryogenesis and at hatching did not differ significantly. However, all haploids died within next few days after hatching. Cytogenetic analysis of the androgenetic embryos confirmed their haploid status. Moreover, apart from the intact paternal chromosomes, residues of the irradiated maternal chromosomes observed as chromosome fragments were identified in some of the haploids. Provided results suggested that rainbow trout X and Y chromosomes despite morphological and genetic differences are at the early stage of differentiation and still share genetic information responsible for the proper embryonic development.

An update to the chromosomal data of the European bitterling Rhodeus amarus (Teleostei: Cypriniformes: Acheilognathinae)

Chromosomal abnormalities in secondary bovine oocytes matured in vitro up to 48 hours Abstract Chromosomal abnormalities in secondary bovine oocytes matured in vitro up to 48 hours. 21st International Colloquium on Animal Cytogenetics and Gene Mapping Rubessa M., Pauciullo A., Peretti V., Iannuzzi L., Ramunno L., Di Berardino D.Edited by: L. Iannuzzi, A. Perucatti, A. Iannuzzi, A. Pauciullo, V. Genualdo, D. Incarnato, L. Keller (CNRISPAAM, Naples, Italy)Sister Chromatid exchange (SCE) test in river buffalo cells treated with Furocoumarins. / Iannuzzi A.; Perucatti A.; Genualdo V.; Pauciullo A.; Pucciarelli L.; Incarnato D.; Melis R.; Porqueddu C.; Marchetti M.; Iannuzzi L.. In: CHROMOSOME RESEARCH. ISSN 0967-3849. 22(2014), pp. 421-421. Original Citation: Sister Chromatid exchange (SCE) test in river buffalo cells treated with Furocoumarins.Comparative FISH-mapping of TNF, STAT5A and MNTR1A fecundity genes on river buffalo, cattle, sheep and goat. / Iannuzzi A.; Perucatti A.; Pauciullo A.; Genualdo V.; Incarnato D.; Pucciarelli L.; De Lorenzi L.; Parma P.; Iannuzzi L.. In: CHROMOSOME RESEARCH. ISSN 0967-3849. 22(2014), pp. 418-418. Original Citation: Comparative FISH-mapping of TNF, STAT5A and MNTR1A fecundity genes on river buffalo, cattle, sheep and goat.Multicolor FISH with 10 specific painting probes for the rapid identification of the sub-metacentric river buffalo autosomes (Bubalus bubalis, 2n=50) / Pauciullo A.; Perucatti A.; Iannuzzi A.; Incarnato D.; Genualdo V.; Pucciarelli L.; Di Berardino D.; Iannuzzi L.. In: CHROMOSOME RESEARCH. ISSN 0967-3849. 22(2014), pp. 410-410. Original Citation: Multicolor FISH with 10 specific painting probes for the rapid identification of the sub-metacentric river buffalo autosomes (Bubalus bubalis, 2n=50)

Developmental competence of eggs produced by rainbow trout Doubled Haploids (DHs) and generation of the clonal lines

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.