Dimethyl acetamide and dimethyl sulfoxide associated at glucose and egg yolk for cryopreservation of Pseudoplatystoma corruscans semen

This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.(AU)

Este estudo teve como objetivo desenvolver um protocolo para a criopreservação do sêmen de Pseudoplatystoma corruscans. Para tal, machos maduros foram induzidos hormonalmente com uma dose única de extrato de hipófise de carpa (5 mg/kg de peso vivo). O sêmen foi coletado e avaliado. Sendo testados para compor os diluentes, dois crioprotetores: dimetil acetamida (DMA) e dimetil sulfóxido (Me2SO), em duas concentrações (8% e 10%), + 5,0% glicose + 10% gema de ovo. O sêmen foi diluído na proporção 1: 4 (sêmen: extensor), embalado em palhetas de 0,5 mL e congelado em container dryshipper em vapores de nitrogênio líquido. Após o descongelamento, foram avaliados os aspectos cinéticos espermáticos, a morfologia espermática e a integridade do DNA dos espermatozoides criopreservados. Os machos de P. corruscans produziram sêmen com motilidade espermática > 80%. Todos os tratamentos proporcionaram após o descongelamento sêmen com motilidade espermática total > 40%, sem diferença significativa (P < 0,05) entre eles, como também entre os demais parâmetros cinéticos espermáticos avaliados. Os tratamentos com DMA proporcionaram uma menor fragmentação do DNA dos gametas. Malformações espermáticas foram identificadas, tanto no sêmen fresco, como no criopreservado, sendo identificado um aumento discreto dessas malformações nos espermatozoides das amostras de sêmen descongeladas de P. corruscans.(AU)

Gonadal morphology and difference in reproductive development of two isolated populations of Astyanax rivularis (Teleostei, Characidae).

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.

Preliminary study on testicular germ cell isolation and transplantation in an endangered endemic species Brycon orbignyanus (Characiformes: Characidae).

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 influence of increased water temperature on the duration of spermatogenesis in a neotropical fish, Astyanax altiparanae (Characiformes, Characidae).

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 Aund, Adiff, 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.

Sperm antioxidant system in ocellate river stingray Potamotrygon motoro at transition from seminal vesicle to cloaca.

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.

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.

Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol

This study aimed to determine the semen characteristics of Astyanax lacustris after hormonal induction and to evaluate the sensitivity of the species sperm to cryoprotective solutions based on the cryoprotectants dimethyl sulfoxide and methyl glycol. Volume, color, sperm concentration, total motility and aspects of sperm movement were analyzed using "Integrated Semen Analysis System". Three different extenders were tested: A) glucose 5%+egg yolk 10%, B) BTS®5% and C) glucose 5% and two permeable cryoprotectants: dimethyl sulfoxide (Me2SO) and methyl glycol (MTG). Fresh A. lacustris semen presented total motility of 76.6±11.2%, motility duration of 33.0±2.2s, sperm concentration of 7.22±3.2×109sptz/mL and seminal osmolality of 219±0.03mOsm/kg-1. The toxicity test showed the highest total motility values at the MTG15%+A, Me2SO15%+B and Me2SO10%+C dilutions, and the Me2SO10%+C and Me2SO15%+C dilutions presented the highest values for curvilinear velocity, linear velocity and average velocity. The tested protocol was not effective at maintaining the viability of A. lacustris semen after freezing because no motility was observed in any of the dilutions. However, the Comet Assay demonstrated that cryoprotectant solutions were effective in protecting the genetic material of cells, as DNA damage levels were low, with no difference between control and Me2SO10% + A, dilutions MTG10%+C, Me2SO10%+B and Me2SO15%+B.(AU)

O objetivo deste estudo foi determinar as características do sêmen de Astyanax lacustris após indução hormonal e avaliar a sensibilidade dos espermatozoides da espécie a soluções crioprotetoras baseadas nos crioprotetores dimetilsulfóxido e metilglicol. Volume, cor, concentração espermática, motilidade total e aspectos do movimento espermático foram analisados usando o "Sistema Integrado de Análise de Sêmen (ISAS®CASA)". Três extensores diferentes foram testados: A) glicose 5%+gema de ovo 10%, B) BTS® 5% e C) glicose 5% e dois crioprotetores permeáveis: dimetilsulfóxido (Me2SO) e metilglicol (MTG). O sêmen fresco de A. lacustris apresentou motilidade total 76,6±11,2%, duração da motilidade 33,0±2,2s, concentração de espermatozoides 7,22±3,2×109sptz/mL e osmolalidade seminal 219±0,03mOsm/kg-1. O teste de toxicidade apresentou maiores valores de motilidade total nas diluições MTG15%+A, Me2SO15%+B e Me2SO10%+C, e as diluições Me2SO10%+C e Me2SO15%+C apresentaram maiores valores de velocidade curvilínea, velocidade linear e velocidade média. O protocolo testado não foi eficaz em manter a viabilidade do sêmen de A. lacustris pós-congelamento, pois não foi observada motilidade em nenhuma das diluições. No entanto, o Ensaio Cometa demonstrou que as soluções crioprotetoras eram eficazes na proteção do material genético das células, pois os níveis de dano ao DNA eram baixos, sem diferença entre controle e Me2SO10%+A, MTG10%+C, Me2SO10%+B e Me2SO15%+B.(AU)

Sperm motility and lipid composition in internally fertilizing ocellate river stingray Potamotrygon motoro.

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 ±â€¯1% saturated FAs, 28 ±â€¯1% monounsaturated FAs (MUFAs), and 41 ±â€¯1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ±â€¯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.

Morphological evaluation of Prochilodus lineatus embryos after vitrification-thawing in high-osmolarity cryoprotectant solution.

We aimed to vitrify embryos of Prochilodus lineatus in a high-osmolarity cryoprotectant solution, evaluating, after the vitrification-thawing process, their morphological changes. Thus, 240 embryos in the 20-somite phase (20S) were exposed for 20 min to one main internal cryoprotectant solution (1,2-propanediol-PROP), divided into four immersion sequence steps of five minutes each. The first three steps were performed in solutions containing only a main internal cryoprotectant (PROP-2, 3 and 4 M), and the fourth step in a high-osmolarity solution combining internal (PROP + dimethyl sulphoxide-Me2 SO) and external cryoprotectants (sucrose-SUC). The final concentration of vitrification was PROP 5 M + Me2 SO 5 M + SUC 0.2 M. During vitrification, the straws exhibited a translucent solid appearance; however, during thawing, their structure became totally opaque and white. After thawing, the embryos suffered an increase in volume and presented morphological changes including protrusions on the surface of the yolk sac, yolk sac rupture, and optical vesicle degradation. On the inside, we observed intercellular spaces and a yolk syncytial layer (YSL) with altered chromatin. Yet, structures such as somites, neural tube, endoderm and epidermis presented cells with a nucleus and integral mitochondria. We conclude that the use of the tested cryoprotectant solution permits the formation of a vitreous solid and preserves part of the cells of the blastoderm. Yet, the heating protocol does not control recrystallization, resulting in the formation of serious morphological anomalies that prevent the preservation of the embryonic unit.

Ovarian cycle in Devario aequipinnatus with emphasis on oogenesis.

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.

Embryonic development and larval growth of Brycon nattereri Günther, 1864 (Characidae) and its implications for captive rearing.

The aim of this study was to describe, for the first time, the embryogenesis and larval growth of the Paraitinga Brycon nattereri Günther, 1864 reared in captivity. After artificial fertilization, eggs were incubated at constant temperature (~19°C) and collected every 15 min during the first 3 h and then every 3 h until hatching. Five larvae were collected daily over 15 days for evaluation of the length, yolk sac volume and specific growth rate. The following stages of embryonic development were identified: zygote, cleavage, gastrula, segmentation and larval. The hatching occurred after 50-54 h, with larvae poorly developed and fully depigmented, devoid of mouth and swimming capacity, presenting 6.32 mm total length and 3.64 mm3 yolk sac volume. The mouth opening was observed between days 3-4 after hatching. The yolk sac absorption was slow during the first 3 days, increasing sharply after this period, being completed on the day 11. During this period there was a decrease in the larval growth rate. After yolk sac absorption, an increase in the growth rate was observed that coincided with the start of exogenous feeding. Cannibalism was not observed during the 15 days of evaluation. The initial development of B. nattereri was slow and poorly developed larvae in relation to other Brycon species, certainly due to the lower temperature required for egg incubation and larval rearing. Other studies are needed in order to develop techniques to improve the methods of incubating eggs and feeding larvae.

The efficiency of spermatogenesis and the support capacity of Sertoli cells in Characiformes.

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.

Effects of cryoprotectants and low temperatures on hatching and abnormal embryo development of Prochilodus lineatus (Characiformes: Prochilodontidae)

This study evaluated the effect of the cryoprotectants and the low temperatures on the embryonic development of Prochilodus lineatus, describing their main morphological alterations. On chilling sensitivity test, the survival rates at the twenty somites stage (20S) were 53.6% at 0ºC, and 100% in 5ºC. To test toxicity, the embryos were exposed to a graded series of 1,2-Propanediol (PROP), dimethyl sulfoxide (Me2SO4) and glycerol (GLY), terminating in a solution of high osmolarity. There was no significant difference in the embryos survival of toxicity test between series of PROP and Me2SO4 in the 6S and 20S. In the cooling protocols, were evaluated the effects of low temperature associated with cryoprotectants. At 5ºC, PROP showed survival rates above 75% in the gastrula stage (G) and above 90% in the 6S and 20S stages. High rates of abnormalities were observed, and the most recurrent were: small bodies, fins presenting uncontrolled cell growth, membrane rupture, and retraction. These results demonstrate the need to use cryoprotectant solutions, even when there is no ice nucleation, and, on the other hand, shows that high cryoprotectant concentrations promote numerous morphological lesions, compromising normal embryonic development.(AU)

Este estudo avaliou os efeitos dos crioprotetores e das baixas temperaturas no desenvolvimento embrionário de Prochilodus lineatus, descrevendo suas principais alterações morfológicas. Nos testes de sensibilidade ao frio, as taxas de sobrevivência, no estágio de vinte somitos (20S), foram de 53,6% a 0ºC e 100% a 5ºC. Nos testes de toxicidade, os embriões foram expostos a uma série graduada dos crioprotetores: 1,2-Propanediol (PROP), dimetilsulfóxido (Me2SO4) e glicerol (GLY), terminando em uma combinação de alta osmolaridade. Não houve diferença significativa na sobrevivência dos embriões nas séries do PROP e Me2SO4 nos estágios de 6S e 20S. Nos protocolos de resfriamento, foram avaliados os efeitos da baixa temperatura associados as séries crioprotetoras. A 5ºC, o PROP apresentou taxas de sobrevivência acima de 75% no estádio de gástrula (G) e acima de 90% nos estádios 6S e 20S. Foram observadas altas taxas de anormalidades sendo as mais recorrentes: corpos pequenos, nadadeiras apresentando crescimento celular descontrolado, ruptura da membrana e retração vitelínica. Estes resultados demonstram a necessidade do uso das soluções crioprotetoras, mesmo não havendo nucleação de gelo e, em contrapartida, demonstra que as elevadas concentrações crioprotetoras promovem numerosas lesões morfológicas, comprometendo o desenvolvimento embrionário.(AU)

Hormonal induction of Brycon cephalus (Characiformes, Characidae) to spermiation using D-ala6, pro9net-mGnRH + metoclopramide.

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.

Oogenesis in Laetacara araguaiae (Ottoni and Costa, 2009) (Labriformes: Cichlidae).

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.

The effects of temperature and busulfan (Myleran) on the yellowtail tetra Astyanax altiparanae (Pisces, Characiformes) spermatogenesis.

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.

Morphology of the urogenital papilla and its component ducts in Astyanax altiparanae Garutti & Britski, 2000 (Characiformes: Characidae)

The histological description of the urogenital papilla is an important tool to comprehension of the reproductive mechanisms in fish, as well as a pre-requisite to germ cell transplantation in adult fish, besides to be a good biological indicator to environmental changes. Was performed the histological description of the urogenital papilla and its component ducts in the tetra Astyanax altiparanae. The genital and urinay ducts pass separately throughout most part of its extension, joining in a single duct before opening. In males this opening is asymmetric and seems to have double origin, being completely surrounded by striated muscle fibers, while in females it is symmetric and the muscle fibers does not surround it totally. Spermatic duct and oviduct undergo changes throughout their extension, mainly in the morphology of the surrounding epithelium. In the spermatic duct, squamous epithelial cells change to columnar and cuboid with possible secretory activity, close to testes. In the oviduct, anteriorly epithelial cells are also squamous, however, close to ovary there are lamellae composed by a pseudostratified epithelium with columnar and cuboid cells. The urinary duct is highly similar for both sexes presenting globoid cells, which description is known in mammals, however, rare in fish.

A descrição histológica da papila urogenital é uma importante ferramenta para a compreensão dos mecanismos reprodutivos em peixes, assim como um pré-requisito para a realização do transplante de células germinativas em peixes adultos, além de um bom indicador biológico de possíveis alterações ambientais. Foi realizada a investigação histológica da papila urogenital e seus ductos constituintes no lambari Astyanax altiparanae. Os ductos genital e urinário ocorrem separadamente ao longo de maior parte de sua extensão, entretanto, unem-se em um ducto simples antes de abrir para o meio externo. Nos machos esta abertura é assimétrica e parece ter dupla origem, sendo completamente envolvida por fibras musculares estriadas, enquanto nas fêmeas ela é simétrica e as fibras musculares não a envolve totalmente. O ducto espermático e oviducto sofrem alterações ao longo de sua extensão, principalmente na morfologia do epitélio que os envolve. No ducto espermático as células epiteliais passam de pavimentosas a colunares e cuboides, com possível atividade secretora, à medida que se aproxima dos testículos. No oviducto, anteriormente as células também são epiteliais pavimentosas, entretanto, próximo aos ovários, formam-se lamelas compostas por um epitélio pseudoestratificado composto por células cuboides e colunares. O ducto urinário é bastante similar em ambos os sexos apresentando células globosas, cuja descrição é conhecida em mamíferos, porém rara em peixes.

Structural analysis of embryogenesis of Leiarius marmoratus (Siluriformes: Pimelodidae).

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.

Histochemical and morphological features of biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga, Brycon nattereri (Characiformes).

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 ± 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 µm in thickness with eight pore canals/µm2. Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Endohelminths in Cichla piquiti (Perciformes, Cichlidae) from the Paraná river, São Paulo state, Brazil / Endohelmintos de Cichla piquiti (Perciformes, Cichlidae) procedentes do rio Paraná, estado de São Paulo, Brasil

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.

Cinquenta espécimes de Cichla piquiti foram colhidos do Rio Paraná, a jusante da Usina Hidrelétrica de Ilha Solteira, Brasil, na pesquisa de endohelmintos. Todos os peixes estavam parasitados por, pelo menos, uma espécie de helminto (prevalência total [P] = 100%). Foram encontrados oito taxa de parasitas: os nematóides Procamallanus (Procamallanus) peraccuratus no intestino; larvas de terceiro estádio dos anisaquídeos Contracaecum sp. e Hysterothylacium sp. na cavidade abdominal, mesentério e serosa do estômago e do intestino, e sobre o fígado e baço; metacercárias do trematódeo Austrodiplostomum compactum no olho e um indivíduo adulto de Genarchella genarchella no estômago; os cestóides Proteocephalus macrophallus, Proteocephalus microscopicus e Sciadocephalus megalodiscus no intestino. Larvas de Hysterothylacium sp. (P = 86%) e P. microscopicus (P = 74%) foram os parasitas mais prevalentes. Anisaquídeos apresentaram maior prevalência e abundância no período seco. Foi observada uma correlação negativa entre as abundâncias de Hysterothylacium sp. e P. microscopicus, sugerindo uma relação de antagonismo/competição entre esses parasitas. Cichla piquiti é descrito como novo hospedeiro para quatro espécies de parasitas. Estes novos registros aumentam significativamente a lista de parasitas de C. piquiti, contribuindo para o conhecimento da relação parasito-hospedeiro e distribuição geográfica desses helmintos.