Attempts for increasing Astyanax altiparanae spawning rates and percentage of responsive oocytes.

In this study, we aimed to propose changes in the protocol of cultured Astyanax altiparanae hypophysation to increase the maximum ovulation rate of 60% registered previously. To that two consecutive experiments were conducted. In the first experiment, three carp pituitary homogenate (CPH) doses (3, 6, and 9 mg/kg) were administered in a single injection, while in the second experiment, the 6 mg/kg CPH dose was tested either in single or double injections. In the first experiment, a single injection of 3 mg/kg CPH did not induce final oocyte maturation or spawning, while a dose of 6 mg/kg CPH resulted in an increase in the plasma level of prostaglandin (PGF2α) at ovulation. The single higher dose of 9 mg/kg CPH did not improve reproductive performance and even though anticipated the resumption of meiosis it was detrimental to the spawning rate. In the second experiment, the dose of 6 mg/kg CPH fractionated into two injections led to a higher spawning rate, spawning volume per female body mass, frequency of post-ovulatory complexes, and PGF2α concentration at ovulation compared to the single injection. The most effective treatment remained the 6 mg/kg of CPH fractionated into two injections, but still providing very low proportion of ovulated females (∼40 %). Overall, this study indicates that the spawning protocols for this species need to be improved to induce ovulation in a larger number of females and be more potent in those females that respond positively.

Morphological analysis of the digestive tract of Hypophthalmus marginatus (Valenciennes, 1840).

The mapará, Hypophthalmus marginatus, is a rheophilic and planktophagous catfish and it is one of the main fishing resources in the Amazon region. To understand its nutritional characteristics, this work described the morphology and histochemistry of the digestive tract of H. marginatus. The oropharyngeal cavity has abundant, long and thin gill rakers, responsible for retaining plankton, while the short and muscular oesophagus promotes the transport of these items to the stomach and prevents water ingestion. The stratified oesophageal epithelium has goblet cells with mucins predominantly neutral that could facilitate the food passage the food passage. The U-shaped siphonal stomach shows a columnar epithelium with neutral mucins to protect against autodigestion. The cardiac and fundic regions have gastric glands, while the pyloric region has a thick muscular layer with a sphincter. The coiled intestine with intestinal quotient of 2.14 ± 0.5 shows long longitudinal folds in the anterior region, decreasing in height in the aboral direction, and highlighting its function in digestion and nutrient absorption. Goblet cells are abundant in posterior intestine and rectum, and rectum shows epithelial cells with mucins in the apical cytoplasm, for protection and defecation. Intraepithelial lymphocytes also are abundant in posterior intestine and rectum, and it acts in immunological defence.

Wild Leporinus friderici induced spawning with different dose of mGnRHa and metoclopramide or carp pituitary extract.

Breeding technology is of utmost importance for reproduction of wild fish in captivity for the reintroduction and selective breeding programs purposes. The main challenge is that when applied to wild undomesticated specimens, conventional protocols often cause breeders and/or embryo mortality and spawning failure. In this study, we evaluated the reproductive performance of wild Leporinus friderici, a great importance fish for subsistence fishing in South American rivers, applying conventional and lower-dose hormonal therapies by means of two consecutive experiments. In the first, a conventional (0.5 and 5.5 mg/kg) and a lower carp pituitary extract (CPE) dose (0.5 and 1.0 mg/kg) were applied. In the second, a conventional mammalian GnRH analogue associated with metoclopramide (mGnRHa + MET) (40 µg mGnRHa + 20 mg MET/kg) and a lower dose (4 µg mGnRHa + 2 mg MET/kg and 8 µg + 4 mg of mGnRHa + MET/kg) were applied. Ovulation was observed in all treatments, however, only lower CPE protocol provided viable embryos. High levels of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and 17ß estradiol (E2) detected in conventional, but not in lower CPE dose, at ovulation, might be associated to the mortality of the embryos. The use of lower CPE dose applied here was the best way to obtain L. friderici viable embryos. These results directly contribute to the knowledge about poorly explored effects of reproductive management and hormonal therapies in wild-type breeders, showing that the use of reduced doses may be an alternative to reproductive success.

Leporinus elongatus induced spawning using carp pituitary extract or mammalian GnRH analogue combined with dopamine receptor antagonists.

Several studies have been developed to support the replacement of the crude carp pituitary extract (CPE) by synthetic products for induced reproduction of South American rheophilic species. However, results have been quite heterogeneous and there is no consensus or a routine use of synthetic products in these species. Thus, the aim of this study was to evaluate the ovulatory process in L. elongatus using different protocols of hormonal induction. Thus, fifteen wild mature females maintained at the Experimental Fish Station, Salto Grande, SP, Brazil were submitted to three different hormonal treatments: CPE (fractioned dose: 0.5 and 5.0 mg kg-1); mGnRHa (single dose: 3.5 µg kg-1) and mGnRHa (single dose: 5.0 µg kg-1). The spawning rate and absolute fecundity were similar among the treatments, but fertility rates were higher for CPE treatment (23.60 ± 9.40) then for mGnRHa treatments (close to or zero zero). Although females ovulated in all treatments, none of them provided viable embryos, showing hatching rates close to zero or zero. Both mGnRHa treatments were more potent for inducing the ovulatory process then CPE treatment, which was evidenced by the fact that the formers showed higher volume density of postovulatory follicles (POF). Accordingly, E2 and 17α-OHP plasma levels were higher for the mGnRHa treated females compared to the CPE one at the time of ovulation. In this study we confirmed previous scientific evidence that, regardless of whether promoting ovulation, the use of conventional CPE and GnRH doses are not appropriate for some South American migratory species, due to the non-attainment of viable embryos. Moreover, we have brought new information about the relationship between reproductive performance and gonadal steroids concentrations using different hormonal therapies, contributing to understand the reasons for Leporinus elongatus embryo loss in induced spawning.

Estrogenic and anti-androgenic effects of the herbicide tebuthiuron in male Nile tilapia (Oreochromis niloticus).

Tebuthiuron is a phenylurea herbicide widely used in agriculture that can reach the aquatic environments, possibly posing negative effects to the aquatic biota. Phenylurea herbicides, such as diuron, are known to cause estrogenic and anti-androgenic effects in fish, but no such effects were yet reported for tebuthiuron exposure. Thus, the aim of this study was to evaluate if tebuthiuron, at environmentally relevant concentrations (100 and 200ng/L) and after 25days of exposure have estrogenic and/or anti-androgenic effects on male of Nile tilapia (Oreochromis niloticus), through the evaluation of plasmatic testosterone (T) and estradiol (E2) levels, brain aromatase (CYP19) levels (western-blot), and by evaluating the histology of the testicles. When compared to the control group, plasmatic T levels decreased about 76% in the animals exposed to 200ng/L of tebuthiuron, while E2 levels increased about 94%, which could be related to a significant increase (77%) in CYP19A1 levels, an enzyme that catalyzes the conversion of androgens into estrogens. Histological analyses of the testicles also demonstrated that tebuthiuron at both tested concentrations caused a decrease in the diameter of the seminiferous tubules and in the diameter of the lumen. Therefore, the gonadosomatic index (GSI) was reduced by 36% % in the animals exposed 200ng/L to tebuthiuron. Indeed, the relative frequency of spermatocytes and spermatids increased respectively 73% (200ng/L) and 61% (100ng/L) in the tebuthiuron exposed animals, possibly due to the impairment of sperm release into the lumen, that was decreased 93% (200ng/L) in the treated animals compared to the control. These results confirm that tebuthiuron causes estrogenic and anti-androgenic effects in Nile tilapias at environmentally relevant concentrations.

Spermatogonia, Germline Cells, and Testicular Organization in the Characiform Prochilodus lineatus Studied Using Histological, Stereological, and Morphometric Approaches.

Prochilodus lineatus is an important representative of the order Characiformes and a species that offers great advantages to fish farming. Therefore, detailed knowledge of its reproductive biology can be applied to various fields of production and biotechnology. In this study, we have identified testicular germ cells during spermatogenesis and have evaluated the volumetric proportion of the testes occupied by structures of the tubular and intertubular compartments. In addition, the individual volume of type A spermatogonia was measured and used to estimate the mean number of these cells per testis. Gonads of adult P. lineatus males were extracted and fixed. Light and transmission electron microscopy were applied to fragments of three testicular regions. Histological, stereological, and morphometric analyses were performed. The stereological data suggest that components of the tubular and intertubular compartments of the P. lineatus testes present a uniform distribution in all three regions and therefore reflect regions with similar distributions of cell types. In addition, P. lineatus testes showed ∼0.6% of type A spermatogonia, as well as a predominance of cysts of primary spermatocytes and spermatids during the reproductive phase evaluated. The results from this study provide a better understanding of the morphology and structure of the testis and of the characterization of the type A spermatogonia in P. lineatus. The nuclear diameter of germ cells also decreases significantly during spermatogenesis. The data presented herein are the first of its kind for the order Characiformes and may be useful for future biotechnology studies on fish reproduction. Anat Rec, 300:589-599, 2017. © 2016 Wiley Periodicals, Inc.

Estrogenic activities of diuron metabolites in female Nile tilapia (Oreochromis niloticus).

Some endocrine disrupting chemicals (EDCs) can alter the estrogenic activities of the organism by directly interacting with estrogen receptors (ER) or indirectly through the hypothalamus-pituitary-gonadal axis. Recent studies in male Nile tilapia (Oreochromis niloticus) indicated that diuron may have anti-androgenic activity augmented by biotransformation. In this study, the effects of diuron and three of its metabolites were evaluated in female tilapia. Sexually mature female fish were exposed for 25 days to diuron, as well as to its metabolites 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU), at concentrations of 100 ng/L. Diuron metabolites caused increases in E2 plasma levels, gonadosomatic indices and in the percentage of final vitellogenic oocytes. Moreover, diuron and its metabolites caused a decrease in germinative cells. Significant differences in plasma concentrations of the estrogen precursor and gonadal regulator17α-hydroxyprogesterone (17α-OHP) were not observed. These results show that diuron metabolites had estrogenic effects potentially mediated through enhanced estradiol biosynthesis and accelerated the ovarian development of O. niloticus females.

Anti-androgenic activities of diuron and its metabolites in male Nile tilapia (Oreochromis niloticus).

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a widely used herbicide which has been frequently detected in surface waters throughout the world. In vivo bioassay guided fractionation studies indicated that diuron may have estrogenic activity augmented by biotransformation. This study evaluated the effects of diuron and three of its metabolites on plasma hormone concentrations and spermatogenesis of the freshwater fish Nile tilapia (Oreochromis niloticus). Sexually mature male fish were exposed for 25 days to diuron, as well to its metabolites 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 3,4-dichlorophenyl-N-methylurea (DCPMU), at concentrations of 200ng/L. Testosterone levels were decreased by diuron, but had limited effects on gonadal histology. Diuron metabolites, however, caused significant decreases in testosterone and in 11-ketotestosterone, gonadosomatic index, diameter of seminiferous tubules and in the mean percentages of germ cells (spermatids and spermatozoa). We conclude that these metabolites have antiandrogenic activity to male Nile tilapia, potentially causing reproductive impairment in male fish.

Dynamics of ovarian maturation during the reproductive cycle of Metynnis maculatus, a reservoir invasive fish species (Teleostei: Characiformes)

In this study, we evaluated the dynamics of ovarian maturation and the spawning processes during the reproductive cycle of Metynnis maculatus. Adult females (n = 36) were collected bimonthly between April 2010 and March 2011. The mean gonadosomatic index (GSI) was determined, ovarian and blood samples were submitted for morphometric evaluation and the steroid plasma concentration was determined by ELISA. This species demonstrated asynchronous ovarian development with multiple spawns. This study revealed that, although defined as a multiple spawning species, the ovaries of M. maculatus have a pattern of development with a predominance of vitellogenesis between April and August and have an intensification in spawning in September; in October, a drop in the mean GSI values occurred, and the highest frequencies of post-ovulatory follicles (POFs) were observed. We observed a positive correlation between the POF and the levels of 17α-hydroxyprogesterone. Metynnis maculatus has the potential to be used as a source of pituitary tissue for the preparation of crude extracts for hormonal induction; the theoretical period for use is from September to December, but specific studies to determine the feasibility of this approach must be conducted.

Neste estudo, avaliamos a dinâmica da maturação ovariana a desova durante o ciclo reprodutivo de Metynnis maculatus. Fêmeas adultas (n = 36) foram coletadas bimestralmente entre abril de 2010 e março de 2011. O índice gonadossomático (IGS) foi calculado e amostras de ovário e de sangue foram submetidas à avaliação morfométrica e das concentrações plasmáticas dos esteroides por ELISA, respectivamente. A espécie apresenta desenvolvimento ovariano assincrônico, com múltiplas desovas. Neste estudo revelamos que mesmo sendo de desova parcelada, os ovários do M. maculatus mostraram um padrão de desenvolvimento com predomínio de atividade vitelogênica entre abril a agosto e intensificação da desova em setembro. Em outubro houve uma diminuição nos valores médios de IGS, bem como registramos as maiores frequências de folículos pós-ovulatórios (FPOs). Observamos uma correlação positiva entre a frequência de FPOs e a concentração plasmática de 17 α-OHP. O M. maculatus tem potencial para ser usado como fonte para uso de hipófise para preparo de extrato bruto para indução hormonal, sendo o período teórico para coleta de hipófises de setembro a outubro, mas estudos específicos para esta finalidade ainda precisam ser desenvolvidos.

Gonadal steroids levels and vitellogenesis in the formation of oocytes in Prochilodus lineatus (Valenciennes) (Teleostei: Characiformes)

The objective of this study was to obtain information about the possible mechanisms related to poor reproductive performance in tropical rheophilic fish. To that effect, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively, for curimbatá (Prochilodus lineatus) breeders. Fish were maintained under experimental conditions for 18 months, and during this period females were randomly sampled every two months for biometric analysis (n=30), blood (n=5/sampling) and ovary (n=5/sampling). After this period EPs females (EPFs) and Cs females (CFs) were submitted to the induced breeding experiments. The results showed that rearing curimbatá for such long time in a cage at this stocking density, reduces its growth, plasma E2 levels and vitellogenesis. During vitellogenesis, the mean plasma estradiollevels of CFs were three times lower than those of EPFs (P<0.01). CFs presented poorer results than EPFs for all the examined parameters of reproductive performance. Taken together these data showed that the reduced estradiol levels during vitellogenesis (and the consequently less intense transition from the previtellogenic to vitellogenic phase) and reduced amounts of yolk are mechanisms associated with the formation of low quality oocytes and shortened and delayed breeding season in this species. Moreover, our data showed that the onset of vitellogenesis (six months before the spawning season) must be considered as a key period related to the formation of oocytes of good quality, and adequate management should be provided throughout the year.

Este estudo teve como objetivo gerar informações básicas sobre os possíveis mecanismos relacionados com os resultados desfavoráveis obtidos com o desempenho reprodutivo em peixes reofílicos tropicais. Para isso tanques-rede (Cs) e viveiros escavados (EPs) foram utilizados como sistemas experimentais para propociar respectivamente condições inadequadas e adequadas para reprodutores de Prochilodus lineatus. Os peixes foram mantidos por 18 meses em viveiros escavados (EPs) e tanques-rede (Cs), e durante este período, foram aleatoriamente coletados a cada dois meses para análise biométrica (n=30), coleta de sangue (n=5/tratamento) e amostragem do ovário (n=5/tratamento). Após este período as fêmeas mantidas em EPs (EPFs) e as fêmeas mantidas em Cs (CFs) foram submetidas aos experimentos de reprodução induzida. Os resultados mostraram que manter curimbatás por este período em Cs, na densidade de estocagem utilizada, reduz seu crescimento, níveis plasmáticos de E2 e vitelogênese. Durante a vitelogênese, os níveis plasmáticos de estradiol das CFs foram três vezes menores do que os das EPFs (P <0,01). As CFs apresentaram resultados inferiores aos das EPFs para todos os parâmetros analisados de desempenho reprodutivo. Em conjunto, estes dados mostram que os níveis reduzidos de estradiol durante a vitelogênese (bem como o consequente atraso na transição da fase previtelogênica para a vitelogênica) e as quantidades reduzidas de vitelo são mecanismos associados com a formação de ovócitos de baixa qualidade; redução e atraso na época de desova nesta espécie. Além disso, nossos dados mostraram que o início da vitelogênese (seis meses antes da época de desova) é um período-chave para formação de ovócitos de boa qualidade, e o manejo adequado deve ser aplicado durante todo o ano.

Testicular structure and description of the seminal pathway in Leporinus macrocephalus (Anostomidae, Teleostei).

The present study examined the testicular structure and the seminal pathway in freshwater fish Leporinus macrocephalus (Garavello and Britski, 1988). Twenty-five specimens of this species were studied. Testicular structure was analyzed using light and transmission electron microscopy. The testicular main ducts were examined by means of conventional histology, corrosion-cast technique and scanning electron microscopy. Additional techniques were applied for polysaccharides histochemistry and immunohistochemistry for androgen receptor. The testicular parenchyma was classified as the anastomosing tubular testis type with spermatogonia occurring along the lengths of the seminiferous tubules. The seminiferous tubules emptied directly into the testicular main ducts. The wall of the testicular main ducts in L. macrocephalus consisted of three layers: epithelium, connective tissue and peritoneum. The epithelium changed from simple cuboidal to pseudostratified. The histochemical analysis revealed the presence of granules PAS positive in the epithelial cells. The immunoreactivity to androgen receptor was noted in the testicular main ducts through all cytoplasmic areas of epithelial cells.

Cell junctions in the germinal epithelium may play an important role in spermatogenesis of the catfish P. fasciatum (Pisces, Siluriformes).

We identified adhesive junctions and gap junctions between Sertoli cells, between Sertoli and germ cells and between germ cells in the testis of P. fasciatum, a catfish of commercial relevance. To investigate the role of these junctions in spermatogenesis, as well as the molecular composition of the junctions, we performed an immunohistochemistry light microscopy as well as an immunogold labelling electron microscopy study with antibodies to adhesive and gap junctions proteins. Testes that were at different stages of spermatogenesis were used. Based on our morphological studies we speculate that Sertoli-germ and germ-germ cell adhesive junctions are important for maintaining the three-dimensional structure of the germinal cysts and an organized arrangement of the germ cells inside the cysts. Connexin 32 was identified in the germ cells and in the cysts walls. Our observations also suggest that Sertoli-germ and germ-germ cells gap junctions may be involved in the mechanism of synchronous development of germ cells.