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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. Sexually mature ocellate river stingrays (Potamotrygon motoro, representative of Chondrichthyes) possess specific features of male gametes: (1) their spermatozoa acquire motility after leaving the testes during the transit through the epididymis; (2) thereafter spermatozoa are stored in a motile state within a specific male organ – the seminal vesicles; and (3) these motile spermatozoa are able to change their motility parameters under the influence of uterine fluid in the female reproductive tract. Based on the changes in these parameters during sperm transit through the male reproductive tract and upon contact with uterine fluid, we have confirmed the occurrence of epididymal sperm maturation and suggested the occurrence of capacitation stage in ocellate river stingrays. [ABSTRACT FROM AUTHOR]

Contents This study aimed to evaluate the effect of five salt solutions in the maintenance of morphological features of cortical alveolus, hydration and fertilization capacity of Prochilodus lineatus oocytes. For this purpose, five saline solutions were tested: Ringer's solution, Ringer's lactate solution, Hank's balanced salt solution ( HBSS), Hank's balanced salt solution without calcium ( HBSS without calcium) and solution for salmonid eggs. Oocytes were maintained for 2 hr in saline solution with controlled temperature subsequently evaluated for hydration, cortical activation and fertilization ability. In the evaluation of the fertilization ability, two controls were used: C1-fertilized oocytes after extrusion-and C2-oocytes kept in ovarian fluid and fertilized after 2 hr. There was a significant reduction in the viability of oocytes C2 (28.8% ± 12.9%) compared to C1 (65.3% ± 26.7%), and no significant differences were found between treatments HBSS and HBSS without calcium and C2. Only HBSS and HBSS without calcium maintained the non-activated state of the gametes, with a fertilization rate of 16.4% ± 6.7% and 5.6% ± 2.3%, respectively; however, they did not extend the viability of oocytes, such that they continued to undergo degradation during the storage period, similar to oocytes retained only in ovarian fluid. [ABSTRACT FROM AUTHOR]

This study aimed to describe testicular and its main ducts structure in the yellowtail tetra Astyanax altiparanae, contributing to the knowledge of the region in which semen is produced, storage and released, focusing mainly on the dynamic of germinal epithelium and Sertoli cells during germ cell maturation. Ten sexually mature male A. altiparanae had their testes processed according to the routine protocols to optical microscopy. Moreover, spermatic ducts and tubular compartment of the testes of three specimens were perfused with vinyl resin for gross anatomy and scanning electron microscopy. Astyanax altiparanae testes are paired organs, separated for most of their extension, joining posteriorly in a spermatic duct formed by a squamous simple epithelium. Seminiferous compartment presents anastomosing tubular type organisation, and spermatogonia spread along its extent. Spermatogenesis is of cystic type, and there is no main testicular duct. Spermatogenesis develops in 'waves', from posterior to anterior part of the gonad. Thus, while sperm is storage posteriorly, spermatogenesis keeps maturing germ cells anteriorly, making the germinal epithelium very dynamic, holding Sertoli cells that change their function as a cystic envelope to produce secretions of the seminal fluid and store sperm. Such kind of development is thought to be responsible by the high prolificacy of this species. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the helminth parasites of Geophagus proximus from the São José dos Dourados River, a tributary of Paraná River, Ilha Solteira Reservoir, São Paulo State, Brazil. From May 2006 to May 2007, 116 G. proximus specimens were examined and seven different taxa of helminth were collected and identified: proteocephalidean plerocercoids (Cestoda); metacercariae of Austrodiplostomumcompactum, Clinostomum heluans and Clinostomum sp. (Trematoda); and Raphidascaris (Sprentascaris) hypostomi, and larvae of Raphidascaris sp. and Contracaecum sp. (Nematoda). All parasites presented the typical aggregated pattern of distribution, as well as the presence of a high number of larval stages, an absence of influence of the host sex and seasonality upon community parameters, as well as a correlation between species richness and host body weight. Moreover, with the exception of A. compactum metacercariae, all helminths found in this study are reported for the first time in G. proximus. [ABSTRACT FROM AUTHOR]

In the Neotropical cichlid Cichla kelberi the spermiogenesis appears to be of the cystic type and is characterized by gradual chromatin condensation in juxtaposed clusters, nuclear rotation (with the flagellum perpendicularly to the nucleus), and consequent nuclear fossa formation. Besides, there is cytoplasma migration to the nuclear base while eliminating the cytoplasmic residual mass. The heads of the spermatozoa are round, with approximately 1.87 μm in diameter; the nucleus has clusters juxtaposed of condensed chromatin. The nuclear fossa in single arc shape is moderate and slightly eccentric to the nuclear base. The midpiece is short and presents a cytoplasmic sheath, which dilated in its extremes. The mitochondria are spherical or slightly elongated. The flagellum presents the classical axoneme 9 + 2 and one pair of fins. The Type I C. kelberi spermatozoon has its flagellum perpendicularly positioned in relation to the nucleus base. The presence of only one flagellum characterize it as the basic spermatozoon in teleost, known as single anacrosomal aquasperm spermatozoon. Besides the patterns of previously cited characteristics between its representatives, the cytoplasmic sheath and flagellum fins presence can be consider as characteristics of the Cichla genus. [ABSTRACT FROM AUTHOR]

In Salminus, spermiogenesis is cystic and gives origin to a type I aquasperm. Spermatid differentiation is characterized by chromatin condensed into thick fibres, nuclear rotation, nuclear fossa formation, cytoplasmic channel formation, mitochondrial fusion producing long and ramified mitochondria, and the presence of several membranous concentric rings around the plasma membrane that encircles the cytoplasmic channel. In Salminus and Brycon, spermatozoa are very similar. They exhibit a spherical nucleus and chromatin condensed into fibre clusters, and a deep nuclear fossa. They show a long midpiece with few elongate mitochondria at the initial region and a cytoplasmic channel completely encircled by one or two membranous concentric rings. The flagellar axis is perpendicular to the nucleus and exhibits the classic axoneme (9 + 2). The very strong similarity observed between Salminus and Brycon spermatozoa supports the hypothesis that these subfamilies are likely to have a monophyletic origin. [ABSTRACT FROM AUTHOR]

Individuals of the same species may present different reproductive tactics depending on the environment in which they develop and mature. The present study aimed to define the gonadal development phases of males and females of Astyanax rivularis and to carry out a comparative analysis of the reproductive development of specimens captured in two isolated environments of the São Francisco River basin in Serra da Canastra, Brazil (Point 1: low vegetation and river showing calm and crystalline waters with small well formations; Point 2: current waters, and well-established areas of arboreal vegetation). Thus, the gonads of A. rivularis specimens were collected, fixed and processed with techniques for light microscopy. Five maturation phases of the females' reproductive cycle were established: immature, developing, spawning capable, regressing and regenerating. Three maturation phases of the males' reproductive cycle were observed: spawning capable, regressing, and regenerating. There are differences in the phases of gonadal development of A. rivularis between the two sampling points so that, possibly, animals upstream of the waterfall demonstrate a delay in the reproductive cycle in relation to animals downstream., (© 2021 Fisheries Society of the British Isles.)

In view of the established climate change scenario and the consequent changes in global temperature, it is essential to study its effects on animal spermatogenesis. Therefore, the aim of this study was to verify the duration of spermatogenesis at different temperatures. For this purpose, 96 male and adult specimens of Astyanax altiparanae were kept in a closed circulation system with water temperature stabilized at 27 °C and 32 °C. Subsequently, the specimens received pulses of BrdU (bromodeoxyuridine) at a concentration of 100 mg/kg/day for 2 consecutive days, and the samples were collected daily for a period of 15 days. Their testes were removed, fixed, processed in historesin, and sectioned in 3 μm, submitted to hematoxylin/eosin staining and to bromodeoxyuridine immunodetection. Partial results of the optimum temperature experiments allowed the classification of A. altiparanae spermatogenic cells in A und , A diff , and type B spermatogonia, spermatocytes, spermatids, and spermatozoa. The duration of spermatogenesis was determined as approximately 6 days for animals at a temperature of 27 °C and 1 day for animals at 32 °C. The elevated temperature was also responsible for increasing cell proliferation, resulting in an increase in the number of spermatocytes, spermatids, spermatozoa, and cell death (cell pyknotic). The duration of spermatogenesis in A. altiparanae was directly affected by the elevated water temperature, causing a reduction in the estimated time of spermatogenesis.

We aimed to develop a simplified protocol for transplantation of Brycon orbignyanus spermatogonial stem cells (SSCs) into Astyanax altiparanae testes. Brycon orbignyanus testes were enzymatically digested and SSC purified by a discontinuous density gradient. Endogenous spermatogenesis was suppressed in A. altiparanae using busulfan or by incubation at 35 °C water, and SSCs from B. orbignyanus labeled with PKH26 were injected into their testes via the urogenital papilla. Twenty-two hours post-transplantation, labeled spermatogonia were observed in A. altiparanae tubular lumen. After 7 days, spermatogonia proliferated in the epithelium, and 21 days post-transplantation, sperm was observed in the lumen. Of surviving host fish, nearly 67% of those treated with busulfan and 85% of those held in warm water showed labeled cells in host germinal epithelium. The present study standardized, by a simple and accessible method, germ cell transplantation between sexually mature Characiformes fish species. This is the first report of xenogenic SSC transplantation in this fish order.

The importance of reactive oxygen species and the antioxidant system in sperm biology has been recognized for different bony fishes but nothing is known in this regard for chondrichthyans. For the first time for cartilaginous fishes, the enzymatic antioxidant system was shown herein to be present in both fractions of sperm (spermatozoa and seminal fluid) collected from two different places (seminal vesicle and cloaca). In internally fertilizing freshwater ocellate river stingray, Potamotrygon motoro, the activity of superoxide dismutase and glutathione peroxidase was not changed upon sperm transition from the seminal vesicle to the cloaca. The activity of catalase was significantly increased for both sperm fractions at transition from the seminal vesicle to the cloaca (1.6 times for spermatozoa and 1.9 times for seminal fluid). The role of the sperm antioxidant system for different aspects of internal fertilization is discussed. The presented results are the initiatory step in uncovering the biochemical events of internal reproduction in Chondrichthyes.

This study reports the occurrence of metacercariae of Austrodiplostomum compactum in Pimelodus platicirris from a Neotropical reservoir in the Grande River, SP, Brazil. A total of 164 fish were collected, of which 12.80% were infected with metacercariae in the eyes. The mean intensity of infection and mean abundance were 1.52±0.14 (1‒3) and 0.23±0.05 (0‒3), respectively. The presence of this parasite with a high intensity of infection can cause exophthalmos, retinal displacement, opacity of the lens, blindness or even death. This is the first record of ocular metacercariae for P. platicirris, thus increasing the number of hosts for A. compactum.

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., Competing Interests: The authors declare no conflict of interest.

All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 ± 1% saturated FAs, 28 ± 1% monounsaturated FAs (MUFAs), and 41 ± 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ± 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

SummaryThis study aimed to understand how germ cell development occurs in females of Devario aequipinnatus, by morphologically describing oogenesis and the reproductive phases. Sexually mature females of D. aequipinnatus (n = 70) were obtained from commercial fisheries and delivered to the Laboratório de Ictiologia Neotropical, UNESP, Ilha Solteira, SP, Brazil. The ovaries were removed, fragmented and fixed following the usual techniques for light microscopy. The stages of ovarian development in D. aequipinnatus begin with the oogonia, which proliferate into new cells or differentiate into prophasic oocytes that, at the end of this process, form the ovarian follicle and end folliculogenesis. In the previtellogenic stage, the oocytes were characterized mainly by the gradual loss of basophilia and an increase in oocyte diameter. Vitellogenesis was marked mainly by the incorporation of yolk granules. Mature oocytes were defined by their migration from the nucleus to the micropyle. Postovulatory follicles and atresic oocytes were also observed. The reproductive phases were classified as: immature, early and final developing, spawning capable, regressing and regenerating. Therefore, the development of an understanding of cell modifications that occurs up to oogenesis is a basic step that is essential for the description of the reproductive biology of D. aequipinnatus, given the lack of information about the reproductive aspects of this species.

This stereological analysis of the types of germ cells and the number of Sertoli cells per cyst in Astyanax altiparanae testes during spermatogenesis is the first such report in Characiformes. Testes of 25 male A. altiparanae were examined. Based on the number of spermatogonia B per cyst (469.2 ± 9.92), we estimated that spermatogonia undergo at least nine mitotic divisions before differentiating into primary spermatocytes. There are four spermatogonia types: undifferentiated spermatogonia A*, undifferentiated spermatogonia, differentiated spermatogonia, and type B spermatogonia. The number of Sertoli cells increased gradually from 1.41 ± 0.51 in the single undifferentiated spermatogonium A* to 9.25 ± 0.50 in cysts of spermatocytes in the leptotene/zygotene stage, possibly related to greater complexity of cellular events during the meiotic stage. The number of germ cells rose dramatically from spermatogonia A (1.0 ± 0) to spermatogonia B (469.2 ± 9.92); however, the quantity of spermatocytes inside the cysts in the leptotene/zygotene stage decreased (300.6 ± 6.97) relative to spermatogonia B, representing a loss of approximately 36% of the former number of cells. This was probably the result of apoptosis, which promotes successful development of the remaining cells during sperm production. The support capacity of Sertoli cells increased gradually during spermatogenesis., (Copyright © 2017. Published by Elsevier Inc.)

We aimed to analyze the oogenesis of adult females of the cichlid fish Laetacara araguaiae. The specimens' gonads were removed and processed for light and transmission electron microscopy. Oogenesis in L. araguaiae showed the following characteristics: a germinal epithelium with three types of oogonia (A-undifferentiated, A-differentiated and B-oogonia), oocytes at meiotic prophase stage and ovarian follicle formation. Oocytes showing primary growth with pre-vitellogenic and cortical alveolus were observed. Similar to data for other cichlids, oocytes in secondary growth or vitellogenesis were characterized by the initial deposition of yolk microgranules. The event that characterizes the maturation stage is nucleolus migration, also called the germinal vesicle, to the oocyte periphery in the direction of the micropyle. The follicular complex undergoes several changes throughout the oocyte stages. To the best of our knowledge this study is the first to describe L. araguaiae oogenesis. Moreover, this study is the first step to better understand the reproductive biology of this species, which shows great potential for use as an ornamental fish.

This study aimed to establish a hormonal induction protocol for spermiation of Brycon cephalus males, using Ala6, Pro9Net-mGnRH + metoclopramide (Ovopel®). Thus, 20 males were used divided into three inductor treatments [⅓ pellet/kg (T1), ⅔ pellet/kg (T2) and 1⅓ pellet/kg (T3)] and one control group (CO), which only received physiological solution applications (0.9% NaCl). All treatments were applied in a single dose. For evaluation of the availability of the treatment, the following seminal parameters were analyzed: seminal volume, subjective spermatic motility, duration of motility, pH, osmolality and spermatic concentration. T3 showed the highest seminal volume (4.66 ± 1.52 ml), and was significantly different in comparison with T1 (2.0 ± 0.9 ml), T2 (3.5 ± 1.3 ml) and CO (2.3 ± 1.2 ml). In relation to spermatic motility, T2 and T3 showed significantly higher levels [5, (81-100%)]. However, T3 showed significantly lower average sperm motility duration than T1, T2 and CO (30 ± 7 s; 28 ± 6 s; 32 ± 8 s, respectively). With regard to the seminal parameters of spermatic concentration, pH and osmolality, no significant variation was verified among treatments. In conclusion, mGnRH + metoclopramide used for hormonal induction of B. cephalus reproduction does not induce changes related to spermatic concentration, pH and osmolality parameters of the seminal fluid and the most adequate doses among tested treatments were ⅔ pellet/kg live fish.

Embryological studies in fish species are useful to the understanding of their biology and systematics. The available biological data in Leiarius marmoratus are scarce and additional information about its reproductive biology is needed, mainly because this species has been commercially exploited and used in production of hybrid lineages. In order to evaluate the temporal-morphological embryonic modifications in L. marmoratus, samples of nearly 200 embryos were collected at random at different stages of development, starting from fecundation (time zero). Embryos were fixed in modified Karnovsk's solution and 2.5% glutaraldehyde, processed and analysed under optic and electron microscopy. The incubation period of L. marmoratus was equal to 14.42 h at a mean temperature of 28.3 ± 0.07°C. The following stages of embryonic development were established: zygote, cleavage, gastrula, organogenesis and hatching. These stages were divided into phases, as follows: cleavage - phases of 2, 4, 8, 16, 32 and 64 cells and morula; gastrula - phases of 25, 50, 75 and 90% of epiboly and blastopore closure; and organogenesis - neurula, segmentation and pre-larval phases. The embryogenesis of L. marmoratus was typical of neotropical teleosteans, with peculiarities in species development.

We aimed to standardize a protocol to suppress spermatogenesis in the characiform fish, Astyanax altiparanae, for future use as a host in germ cell transplant research, opening opportunities for a range of studies, such as spermatogenesis analyses and transgenesis because this species presents livestock characteristics to be used as a biological model. The effects of the chemotherapeutic busulfan (formulated as Myleran), which is used as medicine, therefore not as toxic to humans manipulation as analytical grade busulfan (Fluka) used in previous studies, were evaluated at physiological temperature of 28 °C, ideal for growth and reproduction of A altiparanae, and also at increased temperature 35 °C. The temperature groups were divided into three treatment groups: busulfan, DMSO only, and an untreated control. Macroscopic, histologic, stereological, and ultrastructure analysis showed that, at 28 °C, busulfan did not cause depletion of germ cells in A altiparanae. However, at 35 °C, sterilization was observed 3 weeks after the initial application. Similar results were obtained with maintenance of fish at 35 °C for a longer period with no accompanying Myleran treatment. This procedure allows reduction in stress and lower mortality resulting from manipulation during busulfan injection and is also suitable for mass treatment because large numbers of fish can be incubated in warm water., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fifty specimens of Cichla piquiti were collected from the Paraná River downstream of the Ilha Solteira Hydroelectric Power Station in Brazil and surveyed for endohelminth parasites. All fish were parasitised by at least one helminth species (overall prevalence [P] = 100%). Eight parasite taxa were present: the nematode Procamallanus (Procamallanus) peraccuratus in the intestines; third-stage larvae of the anisakids Contracaecum sp. and Hysterothylacium sp. in the visceral cavity, mesentery and serosa of the stomach and intestines and on the liver and spleen; the trematodes Austrodiplostomum compactum in the eye (metacercariae) and Genarchella genarchella in the stomach; and the cestodes Proteocephalus macrophallus, Proteocephalus microscopicus, and Sciadocephalus megalodiscus in the intestines. Hysterothylacium sp. larvae (P = 86%) and P. microscopicus (P = 74%) were the most prevalent parasites. Anisakids were more prevalent and abundant in the dry season. A negative correlation between the abundances of Hysterothylacium sp. and P. microscopicus was observed, suggesting a competitive/antagonistic relationship between these parasites. Cichla piquiti represents a new host for four parasite species. These new records significantly increase the list of parasites of C. piquiti, contributing to the knowledge of the host-parasite relationship and the geographical distribution of these helminths.

The embryogenesis of Brycon cephalus was established in seven stages: zygote, cleavage, blastula, gastrula, segmentation, larval and hatching, in an incubation period of 11 h (26 degrees C). The zygote phase was observed directly after fertilization and egg hydration. Cleavage began at 0.5 h of incubation and extended up to the morula phase (1.5 h; +100 blastomeres). Cleavage was meroblastic and underwent the following division pattern: the first five divisions were vertical and perpendicular to each other, following the model 2 x 2, 4 x 2, 4 x 4 and 4 x 8. The sixth division was horizontal and occurred at 1.25 h after fertilization, giving rise to two cell layers (4 x 8 x 2) with 64 blastomeres. At the blastula stage (1.25-1.5 h), an irregular space between the blastomeres, the blastocoele, could be detected and the periblast structure initiated. The gastrula (1.75-6.0 h) was characterized by the morphogenetic movements of epiboly, convergence and cell involution, and formation of the embryonic axis. The segmentation stage (7-9 h) comprised the development of somites, the notochord, optic, otic and Kupffer's vesicles, neural tube, primitive intestine and ended with the release of the tail. The larval stage (up to 10 h) was characterized by the presence of 30 somites and growth and elongation of the larvae. At the hatching stage, the embryos presented more than 30 somites and exhibited swimming movements and a soft chorion. The blastomeres presented euchromatic nuclei, indicating a high mitotic activity and many yolk globules in the cytoplasm. The periblast was constituted of a layer with several nuclei and many vesicles, which grew during the epiboly movement.

SummaryWhile 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.