BackgroundTargeted re-sequencing is one of the most powerful and widely used strategies for population genetics studies because it allows an unbiased screening for variation that is suitable for a wide variety of organisms. Examples of studies that require re-sequencing data are evolutionary inferences, epidemiological studies designed to capture rare polymorphisms responsible for complex traits and screenings for mutations in families and small populations with high incidences of specific genetic diseases. Despite the advent of next-generation sequencing technologies, Sanger sequencing is still the most popular approach in population genetics studies because of the widespread availability of automatic sequencers based on capillary electrophoresis and because it is still less prone to sequencing errors, which is critical in population genetics studies. Two popular software applications for re-sequencing studies are Phred-Phrap-Consed-Polyphred, which performs base calling, alignment, graphical edition and genotype calling and DNAsp, which performs a set of population genetics analyses. These independent tools are the start and end points of basic analyses. In between the use of these tools, there is a set of basic but error-prone tasks to be performed with re-sequencing data.ResultsIn order to assist with these intermediate tasks, we developed a pipeline that facilitates data handling typical of re-sequencing studies. Our pipeline: (1) consolidates different outputs produced by distinct Phred-Phrap-Consed contigs sharing a reference sequence; (2) checks for genotyping inconsistencies; (3) reformats genotyping data produced by Polyphred into a matrix of genotypes with individuals as rows and segregating sites as columns; (4) prepares input files for haplotype inferences using the popular software PHASE; and (5) handles PHASE output files that contain only polymorphic sites to reconstruct the inferred haplotypes including polymorphic and monomorphic sites as required by population genetics software for re-sequencing data such as DNAsp.ConclusionWe tested the pipeline in re-sequencing studies of haploid and diploid data in humans, plants, animals and microorganisms and observed that it allowed a substantial decrease in the time required for sequencing analyses, as well as being a more controlled process that eliminates several classes of error that may occur when handling datasets. The pipeline is also useful for investigators using other tools for sequencing and population genetics analyses.

Artificial reproduction and gamete fertilization were evaluated in Salminus hilarii wild and domesticated broodstocks. Wild and domesticated broodstocks were artificially induced to reproduction using a carp pituitary treatment. Four groups were considered: Group 1 (G1), fish caught in the wild maintained for three years in the same conditions as the domesticated broodstocks and spawned naturally; Group 2 (G2), broodstock born and raised in captivity and spawned naturally; Group 3 (G3), wild broodstocks, which were manually stripped for gamete collection and dry fertilization; and Group 4 (G4), domesticated males and females, also manually stripped. Oocytes, eggs, and larvae were sampled at different time intervals throughout embryonic development. Yolk sac absorption occurred approximately 24-29 h after hatching. Twenty-six h after hatching, the larvae mouths opened. Cannibalism was identified just 28-30 h after hatching. There was no morphological difference in embryonic development among all groups. The number of released eggs per gram of female was: G1: 83.3 ± 24.5 and G2: 103.8 ± 37.4; however, the fertilization success was lower in G2 (42.0 ± 6.37 %) compared with G1 (54.7 ± 3.02%) (P = 0.011). Handstripping of oocytes was not successful and the fertilization rate was zero. The reproduction of this species in captivity is viable, but it is necessary to improve broodstock management to enhance fertilization rates and obtain better fingerling production for restocking programs.A reprodução artificial e fertilização dos gametas foram avaliados em reprodutores selvagens e de cativeiro de Salminus hilarii. Reprodutores selvagens e de cativeiro foram induzidos artificialmente à reprodução utilizando hipófise de carpa. Quatros grupos foram considerados: Grupo 1 (G1), peixes capturados na natureza, mantidos por três anos nas mesmas condições de reprodutores de cativeiro e desovados naturalmente; Grupo 2 (G2), reprodutores nascidos e criados em cativeiro e desovados naturalmente; Grupo 3 (G3), reprodutores selvagens que foram extrusados manualmente para a coleta de gametas e fertilização a seco; e Grupo 4 (G4), com machos e fêmeas domesticadas, também extrusados manualmente. Oócitos, ovos e larvas foram amostrados em diferentes intervalos de tempo ao longo do desenvolvimento embrionário. A absorção do saco vitelínico ocorreu aproximadamente 24-29 h após a eclosão. Vinte e seis h após a eclosão, as larvas abriram a boca. O canibalismo foi identificado apenas 28-30 h após a eclosão. Não houve diferença morfológica no desenvolvimento embrionário entre todos os grupos. O número de ovos liberados por grama de fêmea foi: G1: 83,3 ± 24,5 e G2: 103,8 ± 37,4; embora, o sucesso na fertilização tenha sido menor no G2 (42,0 ± 6,37%) em comparação com G1 (54,7 ± 3,02%) (P = 0,011). A extrusão manual dos oócitos não foi bem sucedida e a taxa de fertilização foi zero. A reprodução em cativeiro desta espécie é viável, mas é necessário um melhor manejo dos reprodutores para aumentar as taxas de fertilização, visando a obtenção de u...

Two experiments were performed using the aromatase inhibitor (AI) letrozole (100mg/kg) to promote sex change, from female-to-male, in protogynous dusky grouper. One experiment was performed during the breeding season (spring) and the other at the end of the breeding season (summer). During the spring, AI promoted sex change after 9 weeks and the sperm produced was able to fertilize grouper oocytes. During the summer, the sex change was incomplete; intersex individuals were present and sperm was not released by any of the animals. Sex changed gonads had a lamellar architecture; cysts of spermatocytes and spermatozoa in the lumen of the germinal compartment. In the spring, after 4 weeks, 11ketotestosterone (11KT) levels were higher in the AI than in control fish, and after 9 weeks, coincident with semen release, testosterone levels increased in the AI group, while 11KT returned to the initial levels. Estradiol (E2) levels remained unchanged during the experimental period. Instead of decreasing throughout the period, as in control group, 17 α-OH progesterone levels did not change in the AI-treated fish, resulting in higher values after 9 weeks when compared with control fish. fshβ and lhβ gene expression in the AI animals were lower compared with control fish after 9 weeks. The use of AI was effective to obtain functional males during the breeding season. The increase in androgens, modulated by gonadotropins, triggered the sex change, enabling the development of male germ cells, whereas a decrease in E2 levels was not required to change sex in dusky grouper.

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The potential benefits of dietary long-chain polyunsaturated fatty acids to fish include improved growth, survival and stress resistance, reproduction and regulation of immune function. The metabolism of eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (ARA; 20:4n-6) into short-lived hormones know as eicosanoids play central roles in the regulation of these responses.

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