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1.
Sci Rep ; 9(1): 13861, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554831

RESUMO

Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

2.
PLoS One ; 14(4): e0205481, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30998742

RESUMO

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5-10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.


Assuntos
Carpas , Criopreservação , Espermatogônias , Animais , Dimetil Sulfóxido/farmacologia , Masculino , Espermatogônias/citologia , Espermatogônias/transplante , Fatores de Tempo
3.
Cryobiology ; 87: 78-85, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30716303

RESUMO

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1 °C/min) and short-term storage (-80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Oogônios/fisiologia , Células-Tronco de Oogônios/fisiologia , Animais , Carpas , Sobrevivência Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Feminino , Congelamento , Metanol/farmacologia , Oogônios/citologia , Ovário/citologia , Propilenoglicol/farmacologia , Sacarose/farmacologia , Trealose/farmacologia
4.
Toxicon ; 154: 1-6, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30243795

RESUMO

Eighty cultures from the Novi Sad Cyanobacterial Culture Collection (NSCCC) were screened for toxicity with Artemia salina bioassay and for common cyanobacterial toxins, microcystins/nodularin (MCs/NOD) and saxitoxin (STX), with ELISA assays. The results show that 22.5% (11) of the investigated cyanobacterial cultures in exponential phase exhibited toxicity in the A. salina bioassay and 38.7% (31) produced MCs/NOD and/or STX. However, the findings in the two methods applied were contradictory. Therefore, A. salina bioassay was repeated on 28 cultures in stationary growth phase, which were positive in ELISA assays but not in the initial A. salina bioassay. Seven more cultures exhibited cell-bound toxicity, and only one extracellular toxicity. The observed difference in the toxicity indicates that cyanobacterial growth phase could affect the screening results. The findings also varied depending on the environment from which the cultures originated. In the initial screening via bioassay, 11.8% (6 cultures out of 51) from terrestrial and 17.2% (5 out of 29) from aquatic environment showed cell-bound toxicity. Furthermore, based on the ELISA assay, 31.4% (16) of the cultures from terrestrial ecosystems were positive for the presence of the investigated cyanotoxins, and 51.7% (15) from aquatic ecosystems. Based on all results, more frequent toxin production was observed in cultures originating from aquatic environments. Furthermore, the group of terrestrial cultures that originated from biological loess crusts were basically non-toxic. The discrepancies in the results by two different methods indicates that the use of several complementary methods would help to improve the assessment of cyanobacterial toxicity and cyanotoxin analyses.


Assuntos
Toxinas Bacterianas/toxicidade , Cianobactérias/química , Cianobactérias/citologia , Toxinas Marinhas/toxicidade , Microcistinas/toxicidade , Animais , Artemia/crescimento & desenvolvimento , Cianobactérias/metabolismo , Ecossistema , Sérvia , Testes de Toxicidade/métodos
5.
Reprod Domest Anim ; 53(5): 1253-1258, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29938837

RESUMO

The aim of this study was to optimize the conditions for hypothermic storage of spermatogonial stem cells (SSCs) and oogonial stem cells (OSCs) of common carp Cyprinus carpio. This was conducted by storing gonadal tissue or isolated cells for 24 hr under hypothermic conditions in the first experiment and by testing two different storage media (L-15 or DMEM supplemented with 10% FBS and 25 mM HEPES) and regular medium change (every 4 days) during two weeks of hypothermic storage in the second experiment. During the first 24 hr, isolated cells showed no decrease in viability, while cells obtained from hypothermically stored tissues displayed significantly lower viability after only 6 hr (Tukey's HSD, p < 0.01) indicating that hypothermic storage of isolated cells is superior to storing tissue pieces. The 2-week trial demonstrated that storage media have a profound influence, while regular medium exchange does not have a positive effect on cell viability. Viability of SSCs and OSCs after two weeks was approximately 40% and 25%, respectively; however, survival of ~70% was obtained after 10 days of storage for SSCs and 7 days for OSCs. Hypothermic storage developed in this study has many practical applications during the development of surrogate broodstock technologies for common carp, but also in carp hatcheries and for the conservation of genetic resources of closely related cyprinid species.


Assuntos
Sobrevivência Celular , Criopreservação/métodos , Crioprotetores/química , Células Germinativas/citologia , Animais , Carpas , Separação Celular , Fatores de Tempo
6.
Sci Total Environ ; 635: 1047-1062, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29710560

RESUMO

Cyanobacteria are present in many aquatic ecosystems in Serbia. Lake Ludos, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water, as well as other aquatic organisms and, a survey of historical data on Lake Ludos, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.


Assuntos
Conservação dos Recursos Naturais/métodos , Ecossistema , Lagos/microbiologia , Animais , Cianobactérias , Monitoramento Ambiental , Eutrofização , Peixes , Sérvia
7.
Fish Physiol Biochem ; 44(6): 1487-1498, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29756177

RESUMO

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.


Assuntos
Transplante de Células/veterinária , Embrião não Mamífero/citologia , Células Germinativas/citologia , Células Germinativas/transplante , Oncorhynchus mykiss/embriologia , Salmonidae/embriologia , Transplante Heterólogo/veterinária , Animais , Península Balcânica , Diferenciação Celular , Transplante de Células/métodos , Conservação dos Recursos Naturais , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Oncorhynchus mykiss/genética , Salmonidae/classificação , Salmonidae/genética
8.
Fish Physiol Biochem ; 44(6): 1499-1507, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29779063

RESUMO

Vitrification was applied to the sperm of two endangered fish species of Soca River basin in Slovenia, the Adriatic grayling (Thymallus thymallus) and marble trout (Salmo marmoratus) following testing different cooling devices and vitrifying media. Sperm was collected, diluted in species-specific non-activating media containing cryoprotectants, and vitrified by plunging directly into liquid nitrogen without pre-cooling. Progressive motility, curvilinear velocity, and straightness of fresh and vitrified-warmed sperm were evaluated with computer-assisted sperm analysis (CASA). Fertilization trials were carried out to test the effectiveness of vitrification in the case of grayling. A protocol utilizing a glucose-based extender, 30% cryoprotectants (15% methanol + 15% propylene glycol), 1:1 dilution ratio, and droplets of 2 µl on a Cryotop as cooling device yielded the highest post-thaw motility values for both Adriatic grayling (7.5 ± 6.5%) and marble trout (26.6 ± 15.8%). Viable embryos were produced by fertilizing eggs with vitrified grayling sperm (hatching 13.1 ± 11.7%, control hatching 73.9 ± 10.4%). The vitrification protocol developed in this study can be utilized in the conservation efforts for the two species as an alternative to slow-rate freezing when working in field conditions or when specific equipment necessary for slow-rate freezing is not available.


Assuntos
Criopreservação/veterinária , Espécies em Perigo de Extinção , Salmonidae/fisiologia , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática , Vitrificação , Animais , Crioprotetores/farmacologia , Fertilização , Masculino , Salmonidae/classificação
9.
J Vis Exp ; (133)2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29553568

RESUMO

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.


Assuntos
Criopreservação/métodos , Crioprotetores/uso terapêutico , Espermatogônias/crescimento & desenvolvimento , Animais , Crioprotetores/farmacologia , Masculino , Testículo , Peixe-Zebra
10.
Fish Physiol Biochem ; 44(6): 1435-1442, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29560576

RESUMO

The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n = 5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg-1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0-9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.


Assuntos
Carpas/fisiologia , Potássio/metabolismo , Análise do Sêmen/veterinária , Sêmen/fisiologia , Sódio/metabolismo , Motilidade Espermática , Animais , Concentração de Íons de Hidrogênio , Masculino , Concentração Osmolar
11.
Cryobiology ; 76: 154-157, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28438562

RESUMO

Due to a lack of cryopreservation protocols for fish eggs and embryos, alternative techniques which will enable storage of female genetic resources are crucial for future development of reproduction management in conservation biology and aquaculture. Experiments were conducted to develop an optimal vitrification protocol for cryopreservation of brown trout Salmo trutta juvenile ovarian tissue. Needle immersed vitrification (NIV) method was used where ovaries were pinned on an acupuncture needle, passaged through equilibration and vitrification solutions containing different combinations and concentrations of methanol (MeOH), propylene glycol (PG) and dimethyl sulfoxide (Me2SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me2SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively unaltered perinucleolar follicles. The method developed for brown trout could be applied to the conservation of female genetic resources of other salmonid species, including endangered and endemic species or populations.


Assuntos
Criopreservação/métodos , Preservação de Órgãos/métodos , Ovário , Salmonidae , Animais , Sobrevivência Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Feminino , Metanol/farmacologia , Propilenoglicol/farmacologia , Vitrificação
12.
Gen Comp Endocrinol ; 245: 77-83, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27401260

RESUMO

Experiments were carried out to test the efficiency of cryopreservation of whole testicular tissue in tench Tinca tinca and goldfish Carassius auratus and compare it to cryopreservation of isolated testicular cells. Additionally, effects of three cryoprotectants (dimethyl sulphoxyde - Me2SO, methanol - MeOH and ethylene glycol - EG) at three concentrations (1M, 2M and 3M) on post-thaw cell viability were assessed. Tissue pieces/isolated testicular cells were diluted in cryomedia and cryopreserved by slow-rate freezing (1°C/min to -80°C followed by a plunge into the liquid nitrogen). In both species Me2SO and EG generally yielded higher cryosurvival of early-stage germ cells than MeOH, while spermatozoa of neither species displayed such a pattern. In most cases a 3M>2M>1M viability pattern emerged in both species for both sample types regardless of the cryoprotectant used. Sample type (dissociated testicular cells vs testicular tissue) did not seem to affect viability rates of tench early-stage germ cells and goldfish spermatozoa, while the opposite was observed for tench spermatozoa and goldfish early-stage germ cells. Additionally, through histological analysis we displayed that tissue structure mainly remained unaltered after thawing in goldfish. These results indicate that cryopreservation of whole testicular tissue is indeed a valid alternative method to cryopreservation of dissociated testicular cells. Early-stage germ cells obtained from cryopreserved testis can be further used in different purposes such as transplantation into suitable donors while viable sperm might be used for fertilization when feasible.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Cyprinidae , Preservação do Sêmen/veterinária , Testículo , Animais , Criopreservação/métodos , Fertilização/efeitos dos fármacos , Congelamento , Masculino , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Fatores de Tempo
13.
Gen Comp Endocrinol ; 245: 102-107, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27174751

RESUMO

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.


Assuntos
Anguilla/fisiologia , Criopreservação/veterinária , Percas/fisiologia , Preservação do Sêmen/veterinária , Motilidade Espermática/fisiologia , Animais , Crioprotetores/farmacologia , Fertilização , Masculino , Metanol , Análise do Sêmen , Espermatozoides , Vitrificação
15.
Harmful Algae ; 55: 66-76, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-28073548

RESUMO

Cyanobacteria can produce toxic metabolites known as cyanotoxins. Common and frequently investigated cyanotoxins include microcystins (MCs), nodularin (NOD) and saxitoxins (STXs). During the summer of 2011 extensive cyanobacterial growth was found in several fishponds in Serbia. Sampling of the water and fish (common carp, Cyprinus carpio) was performed. Water samples from 13 fishponds were found to contain saxitoxin, microcystin, and/or nodularin. LC-MS/MS showed that MC-RR was present in samples of fish muscle tissue. Histopathological analyses of fish grown in fishponds with cyanotoxin production showed histopathological damage to liver, kidney, gills, intestines and muscle tissues. This study is among the first so far to report severe hyperplasia of intestinal epithelium and severe degeneration of muscle tissue of fish after cyanobacterial exposure. These findings emphasize the importance of cyanobacterial and cyanotoxin monitoring in fishponds in order to recognize cyanotoxins and their potential effects on fish used for human consumption and, further, on human health.


Assuntos
Toxinas Bacterianas/análise , Toxinas Bacterianas/farmacologia , Carpas , Cianobactérias/fisiologia , Monitoramento Ambiental , Tanques/química , Tanques/microbiologia , Animais , Sistema Digestório/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Rim/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Sérvia , Espectrometria de Massas em Tandem
16.
Artigo em Inglês | MEDLINE | ID: mdl-26023756

RESUMO

Cyanobacteria are present in all aquatic ecosystems throughout the world. They are able to produce toxic secondary metabolites, and microcystins are those most frequently found. Research has displayed a negative influence of microcystins and closely related nodularin on fish, and various histopathological alterations have been observed in many organs of the exposed fish. The aim of this article is to summarize the present knowledge of the impact of microcystins and nodularin on the histology of fish. The observed negative effects of cyanotoxins indicate that cyanobacteria and their toxins are a relevant medical (due to irritation, acute poisoning, tumor promotion, and carcinogenesis), ecotoxicological, and economic problem that may affect both fish and fish consumers including humans.


Assuntos
Carcinogênese/patologia , Cianobactérias/química , Doenças dos Peixes/patologia , Microcistinas/toxicidade , Peptídeos Cíclicos/toxicidade , Animais , Carcinogênese/induzido quimicamente , Doenças dos Peixes/induzido quimicamente , Microcistinas/envenenamento , Peptídeos Cíclicos/envenenamento
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