Sperm Lipid Composition in Early Diverged Fish Species: Internal vs. External Mode of Fertilization

The lipid composition of sperm membranes is crucial for fertilization and differs among species. As the evolution of internal fertilization modes in fishes is not understood, a comparative study of the sperm lipid composition in freshwater representatives of externally and internally fertilizing fishes is needed for a better understanding of taxa-specific relationships between the lipid composition of the sperm membrane and the sperm physiology. The lipidomes of spermatozoa from stingray, a representative of cartilaginous fishes possessing internal fertilization, and sterlet, a representative of chondrostean fishes with external fertilization, have been studied by means of nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray MS, gas chromatography-(GC) MS, and thin-layer chromatography (TLC). NMR experiments revealed higher cholesterol content and the presence of phosphatidylserine in stingray compared to sterlet sperm. Unknown MS signals could be assigned to different glycosphingolipids in sterlet (neutral glycosphingolipid Gal-Cer(d18:1/16:0)) and stingray (acidic glycosphingolipid sulpho-Gal-Cer(d18:1/16:0)). Free fatty acids in sterlet sperm indicate internal energy storage. GC-MS experiments indicated a significant amount of adrenic acid, but only a low amount of docosahexaenoic acid in stingray sperm. In a nutshell, this study provides novel data on sperm lipid composition for freshwater stingray and sterlet possessing different modes of fertilization

Structural and ultrastructural analysis of embryonic development of Prochilodus lineatus (Valenciennes, 1836) (Characiforme ; Prochilodontidae)

This survey was performed to characterize the embryogenesis of Prochilodus lineatus. Seven stages of embryo development were identified - zygote, cleavage, blastula, gastrula, segmentation, larval and hatching - after a period of incubation of 22h (24 degrees C) or 14h (28 degrees C). The following cleavage pattern was identified: the first plane was vertical (2 blastomeres); the second was vertical and perpendicular to the first (4 blastorneres); the third was vertical and parallel to the first (4 x 2); the fourth cleavage was vertical and parallel to the second (4 x 4); the fifth was vertical and parallel to the first (4 x 8); and the sixth cleavage was horizontal (64 blastomeres). At the blastula stage (3.0-4.0 h (24 degrees C); 1.66-2.0h (28 degrees C) irregular spaces were detected and periblast structuring was initiated. At the gastrula stage (4.0-8.0 h (24 degrees C); 3.0-6.0 h (28 degrees C) the epiboly, convergence and cell movements, as well as the formation of embryonic layers, had begun. The segmentation stage (10.0-15.0h (24 degrees C); 7.0-10.0h (28 degrees C)) was characterized by a rudimentary formation of organs and systems (somites, optic vesicle and intestinal delimitation). The embryo at the larval stage (16.0-21.0 h (24 degrees C); 11.0-13.0 h (28 degrees C)) showed a free tail, more than 25 somites, an optic vesicle and a ready-to-hatch larval shape. The blastomeres at cleavage stage had disorganized nuclei indicating high mitotic activity. At gastrula, the blastomeres and the periblast had euchromatic nuclei and a large number of mitochondria and vesicles. The yolk was organized into globose sacs, which were dispersed into small pieces prior to absorption

Seminal analysis, cryogenic preservation, and fertility in matrinxã fish, Brycon cephalus (Günther, 1869)

Visando o desenvolvimento das técnicas de reprodução em peixes, foi realizada a caracterização e a criopreservação do sêmen de Brycon cephalus. As características seminais observadas foram: sêmen de coloração leitosa quase transparente, com um volume médio de 4mL e concentração espermática de 9.617±1.630 x 10(6) espermatozóides/mm³. Os espermatozóides (comprimento = 31,288±4,47 µm) são do tipo aquasperm, apresentando uma pequena cabeça arredondada (comprimento= 1,752±0,18 µm; largura = 1,752±0,17 µm) sem vesícula acrosomal, com um núcleo com cromatina altamente compactada, formando grumos grosseiros e um complexo centriolar localizado na fossa nuclear; uma peça intermediária (comprimento = 2,561±0,44 µm) que se estreita no sentido antero-posterior, um canal citoplasmático e um flagelo (comprimento = 29,521±4,37 µm). Os testes de fertilização com sêmen descongelado demonstraram um efeito significante (alfa = 0,05) no aumento da fertilidade quando utilizado o diluente tipo B, para ambos os tipos de palhetas, 0,5mL e 4,0mL. Não foi constatado efeito significativo (alfa = 0,05) entre a utilização de palhetas de 0,5mL ou de 4,0mL em relação à porcentagem de eclosão.Aiming to improve fish reproduction techniques, the characterization and cryopreservation of semen of Brycon cephalus were performed. The seminal characteristics observed were: an almost transparent, milky semen with a mean volume of 4mL, and sperm concentration of 9.617±1.630 x 10(6) spermatozoa/mm³. Spermatozoa (length = 31.288 ±4.47µm) were of the aquasperm type and displayed a small, round head (length = 1.727 ±0.18 µm; width = 1.752 ±0.17µm) without acrosomal vesicle, nucleus with highly condensed chromatin forming coarse clots and centriolar complex located in the nuclear fossa; a midpiece (length = 2.561±0.44µm), narrowed rearward, with a cytoplasmic canal; and a flagellum (length = 29.521 ±4.37µm). Fertilization tests with thawed semen demonstrated a significant effect (alpha= 0.05) on the increase of thawed semen fertility with diluent type B in both 0.5mL and 4.0 mL straws. No significant effect (alpha= 0.05) on hatching rate was observed in both straw sizes used.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Cryogenic preservation of embryos of Prochilodus lineatus (Valenciennes, 1836) (Characiforme; Prochilodontidae)

While the freezing techniques of mammal embryos have been providing promising results, the cryopreservation of teleostean eggs and embryos have remained unsuccessful up to now. Therefore, this work aimed to develop a procedure of cryogenic preservation of embryos of Prochilodus lineatus and to observe, at both structural and ultrastructural levels, the morphological alterations that took place after the application of freezing/thawing techniques. The embryos at the morula stage could not tolerate exposure to the cryoprotectants ethylene glycol monomethyl ether, propylene glycol monomethyl ether, methanol, dimethyl sulphoxide and propylene glycol, presenting 100% of mortality. Embryos at the 4- to 6-somites stage tolerated exposure to propylene glycol and dimethyl sulphoxide, and the results revealed no significant differences (alpha = 0.05) regarding survival from both treatments. None of the freezing, thawing and hydration protocols was effective on preserving embryo viability. The ultrastructural analyses of frozen and thawed embryos showed that cells from ectoderm, somites, notochord and endoderm were structurally intact, with well preserved nuclei and mitochondria. The yolk globules were able to tolerate the freezing process, but the yolk syncytial layer was unorganized, displaying an electron-dense and compacted appearance, collapsed reticules, nuclei with modified chromatin and ruptures on the plasmatic membrane at the contact zone with endoderm. It might be concluded that the procedures tested for freezing were unable to avoid the formation of intracellular ice crystals, leading to drastic morphological modifications and making P. lineatus embryos unviable