Sperm maturation, as a process through which spermatozoa acquire the ability for motility activation and fertilization, is well‐known for many groups of animals. This process takes place in a taxon‐specific manner in different parts of male reproductive tract. In contrast to most fish with external fertilization, a large number of animals have internal fertilization where transit through the epididymis of the male reproductive tract is necessary for the completion of sperm maturation. Although Chondrichthyes are fishes, they exhibit internal fertilization and possess specific male genital anatomy that resembles mammals. The existence of sperm maturation in the epididymis of cartilaginous fishes has been proposed but has yet to be confirmed. In this study, we used mature ocellate river stingrays Potamotrygon motoro, as a representative of Chondrichthyes, collected during the natural spawning period and evaluated sperm motility parameters during transit through the male reproductive tract, and upon contact with uterine fluid. Our data demonstrate that spermatozoa acquire motility after transit through the epididymis and are stored in a motile state within a specific organ – the seminal vesicles. Moreover, these motile spermatozoa are able to increase their velocity under the influence of uterine fluid in the female reproductive tract.

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.

Summary: The yolk syncytial layer (YSL) has been regarded as one of the main obstacles for a successful cryopreservation of fish embryos. The purpose of this study was to identify and characterize the YSL inProchilodus lineatus, a fish species found in southeastern Brazil and considered a very important fishery resource. Embryos were obtained through artificial breeding by hormonal induction. After fertilization, the eggs were incubated in vertical incubators with a controlled temperature (28 °C). Embryos were collected in several periods of development up to hatching and then fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Morphological analyses were carried out under either light, transmission or scanning electron microscopy. The formation of the YSL inP. lineatusembryos starts at the end of the cleavage stage (morula), mainly at the margin of the blastoderm, and develops along the embryo finally covering the entire yolk mass (late gastrula) and producing a distinct intermediate zone between the yolk and the endodermal cells. The YSL was characterized by the presence of microvilli on the contact region with the yolk endoderm. A cytoplasmic mass, full of mitochondria, vacuoles, ribosomes, endomembrane nets and euchromatic nuclei, indicated a high metabolic activity. This layer is shown as an interface between the yolk and the embryo cells that, besides sustaining and separating the yolk, acts as a structure that makes it available for the embryo. The structural analyses identified no possible barriers to cryoprotectant penetration.

The fish embryonic development comprises the events between the egg fertilization up to larvae hatching, being useful for the identification of viable eggs in productivity and survival studies as well as in raising experiments of several species. The goal of the present study was to characterize the embryonic development of Pimelodus maculatus (Siluriformes; Pimelodidae). The embryogenesis was typical of teleosteans, but with differences in relation to other species such as duration of development, type of blastocoel, moment of somite segmentation among others. Six stages of embryonic development were defined: zygote, cleavage, blastula, gastrula, organogenesis (divided in phases: early segmentation and late segmentation) and hatching with a period of incubation equal to 13 hours at 29 ºC and 17 hours at 25 ºC. The extruded oocytes presented a mean diameter of 812 µm before and 1066 µm after hydration. When fertilized, they presented a yellowish coloration and a gelatinous layer surrounding the chorion. The cleavage pattern is described as: 2; 4; 8 (4x2); 16 (4x4); 32 (4x8) and 64 (2x4x8) blastomeres up to morula phase (+64 cells). It was also possible to observe at this phase, the beginning of the formation of the yolk syncyctial layer (YSL). Afterwards, the blastula and gastrula stages followed. The end of gastrula was characterized by the formation of the yolk plug. Subsequently, the differentiation between cephalic and caudal regions began, along with the embryo elongation, structuring of optic, Kupffer's and otic vesicles besides a previously unidentified structure in the yolk syncyctial layer. The end of this stage is typified by the tail detachment. The late segmentation phase was distinguished by a free tail, presence of more than 30 somites, optic and otic vesicles, development of posterior intestine, pigmentation of cephalic and caudal regions of yolk sac and embryo growth. The recently-hatched larvae presented a primordial digestive tract, quite evident and pigmented eyes, closed mouth, encephalic vesicles and a mean length of 3410 µm.