This study aimed to evaluate the ploidy and survival of larvae resulting from crosses between tetraploid females and diploid males of yellowtail tetra Astyanax altiparanae, both females (three diploids and three tetraploids) and males (n = 3 diploids). Breeders were subjected to hormonal induction with pituitary gland extract from common carp fish (Cyprinus carpio). Females received two doses at concentrations of 0.3 and 3.0 mg/kg −1 body weight and at intervals of 6 h. Males were induced with a single dose of 3.0 mg/kg −1 applied simultaneously with the second dose in females. Oocytes from each diploid and tetraploid female were fertilized with semen from the same male, resulting in two crosses: cross 1 (diploid male and diploid female) and cross 2 (diploid male and tetraploid female). The procedures were performed with separate females (diploid and tetraploid) and diploid males for each repetition (n = 3). For ploidy determination, 60 larvae from each treatment were analyzed using flow cytometry and cytogenetic analyses. As expected, flow cytometry analysis showed that progenies from crosses 1 and 2 presented diploid and triploid individuals, respectively, with a 100% success rate. The same results were confirmed in the cytogenetic analysis, in which the larvae resulting from cross 1 had 50 metaphase chromosomes and those from cross 2 had 75 chromosomes. The oocytes have a slightly ovoid shape at the time of extrusion. Diploid oocytes had a size of 559 ± 20.62 μm and tetraploid of 1025.33 ± 30.91 μm. Statistical differences were observed between eggs from crosses 1 and 2 (P = 0.0130). No significant differences between treatments were observed for survival at the 2-cell stage (P = 0.6174), blastula (P = 0.9717), gastrula (P = 0.5301), somite (P = 0.3811), and hatching (P = 0.0984) stages. In conclusion, our results showed that tetraploid females of the yellowtail tetra A. altiparanae are fertile, present viable gametes after stripping and fertilization using the ‘dry method’, and may be used for mass production of triploids. This is the first report of these procedures within neotropical characins, and which can be applied in other related species of economic importance.

Primordial germ cells transplantation is a unique approach for conservation and reconstitution of endangered fish species. This study aimed to establish techniques to culture dechorionated embryos in different incubation systems and also to determine anaesthetic concentration for fish recipients in the larval stage for subsequent primordial germ cell transplantation. Intact and dechorionated embryos were divided into three incubation systems: (1) a control group with manual replacement of the solution; (2) a closed environment with high oxygen with manual replacement of the solution; and (3) constant solution recirculation. This combination resulted in six treatments. For the evaluation of anaesthetics for larvae, the concentrations evaluated were 19.5 mM, 24.4 mM, 29.3 mM, and 34.2 mM of 2-phenoxyethanol. Anaesthesia concentration and recovery at different stages were evaluated. For transplantation, primordial germ cells of Astyanax altiparanae were transplanted into anaesthetised larvae (1 dph) of Prochilodus lineatus. Better results were obtained in the recirculation system for dechorionated embryos of P. lineatus for hatching (54.18%) and normal morphology (50.06%). The 2-phenoxyethanol anaesthetic with a dose of 29.3 mM resulted in shorter induction times, in addition to the recovery time between 5 and 10 min. By using this anaesthetic concentration at transplantation, GFP-positive cells were seen in two recipients, but the cells did not proliferate. This study established an effective incubation system for the development of the dechorionated embryo and determined an effective anaesthetic concentration for P. lineatus larvae. In addition, micromanipulation and transplantation of primordial germ cells in neotropical species were conducted for the first time.

Triploidization plays an important role in aquaculture and surrogate technologies. In this study, we induced triploidy in the matrinxã fish (Brycon amazonicus) using a heat-shock technique. Embryos at 2 min post fertilization (mpf) were heat shocked at 38°C, 40°C, or 42°C for 2 min. Untreated, intact embryos were used as a control. Survival rates during early development were monitored and ploidy status was confirmed using flow cytometry and nuclear diameter analysis of erythrocytes. The hatching rate reduced with heat-shock treatment, and heat-shock treatments at 42°C resulted in no hatching events. Optimal results were obtained at 40°C with 95% of larvae exhibiting triploidy. Therefore, we report that heat-shock treatments of embryos (2 mpf) at 40°C for 2 min is an effective way to induce triploid individuals in B. amazonicus.

Rivulidae comprises a family of fish largely distributed in Brazil that includes 201 species, of which 125 are considered endangered. This fact emphasizes the need for development of conservation strategies including studies on genetics and reproduction. In this paper, we describe aspects of biology and reproduction of the rivuliid species Hypsolebias sertanejo. We outline the reproductive behaviour of this species under laboratory conditions, analyze ploidy status by flow cytometry, describe reproductive behaviour and performance and test dry and wet incubation of eggs. Although H. sertanejo showed well known patterns of reproductive behaviour, we verified many peculiarities inherent to its reproductive biology. As expected, most individuals were diploid (87.71%), however 14.29% were considered mosaics. Although no sterility was observed within mosaics, infertility of these fish was not fully evaluated. Hatching rate of the eggs collected was very low following both dry and wet incubation (5.04 and 3.79%, respectively). These results provide interesting information regarding the reproductive success of this species, and suggest that chromosomal abnormalities described may reduce the survival of H. sertanejo under natural conditions, limiting the perpetuation of this species, and emphasizing the need for more preservation efforts, including artificial propagation and gene banking.

The aim of this study was to evaluate different post-shock temperatures for tetraploid induction in the yellowtail tetra Astyanax altiparanae. Newly fertilized eggs were divided into four groups, three were submitted to heat shock (40°C for 2 min) at 24 min post-fertilization (mpf) and another group remained without shock (control). Groups submitted to temperature shock were further separated at the following temperatures: 22°C, 26°C and 28°C. Survival among embryonic development was counted and at hatching the ploidy was analyzed by flow cytometry. The results showed that the post-shock temperature affects the parameters analyzed and, therefore, must be considered for optimization of the production of tetraploid in A. altiparanae. Those data are innovative and could be used in future studies of basic biology in this species.

A histological characterization of gonadal development in the tetra Astyanax bimaculatus was performed, aimed at determining its reproductive cycle in streams localized inside the Amazonian forest. Collections were carried out monthly from August 2017 to July 2018 at the Zoobotânica Foundation of Marabá, PA. Collected specimens were weighed and measured, and their gonads and liver were removed and weighed to calculate gonadosomatic and hepatosomatic indexes. Gonads were fixed and treated for routine histology for light microscopy. Materials were stained with toluidine blue and haematoxylin and eosin. The Amazonian A. bimaculatus species presented two reproductive periods in the year, one at the end of the winter season and another during the summer. Females showed an asynchronous development of their oocytes and only two reproductive phases of development were observed during the whole period ‘developing’ and ‘spawning capable’. Males presented cystic spermatogenesis, with an anastomosing tubular testis containing spermatogonia spread along the germinal epithelium (unrestricted spermatogonial). These morphological characteristics are considered phylogenetically more primitive. Male specimens were observed to have five different phases during the period: immature, initial maturation, mid maturation, final maturation and regression. The huge fluctuations in Amazonian streams was observed, in which water volumes varied considerably across seasons, culminating even in total drought. In spite of this, A. bimaculatus could be found during all seasons, showing its impressive reproductive adaptation to its conditions.

In this study we analyzed whether the in vivo storage of oocytes (time after ovulation until fertilization) affects the survival and the ploidy status of the yellowtail tetra Astyanax altiparanae. Fish were induced to spawn and, after ovulation, a small aliquot was stripped and immediately fertilized (positive control group). Subsequently, aliquots (~150 oocytes) were stripped and fertilized at various time points of 60, 120, 180 or 240 min. Developmental stages, abnormalities, survival and the ploidy status of the hatched larvae were examined. As expected, in the control group, 100% of the larvae were diploid. Conversely, triploid individuals were observed just at the 60 min treatment time point (0.6%). In vivo storage of oocytes also influenced the survival rates (P < 0.05); the 180 and 240 min samples, respectively, presented lower survival rates at gastrula (50.10±6.26% and 40.92±5.32%), and somite (17.80±5.14% and 4.41±2.76%) stages and lower hatching rates (12.01±4.04% and 4.41±2.76%). A higher percentage (99.27±0.40%) of normal larvae and only a few abnormal larvae (0.73±0.40%) were observed in the control group (P = 0.0000). This observation did not differ from that observed at the 60 min treatment point (P = 0.9976). A significant increase in the percentage of abnormalities was observed in the other treatments, and, after 240 min, the highest percentage of abnormal larvae was seen (P=0.0024; 83.33±16.67%). In conclusion, we showed that oocyte ageing had a significant effect on survival and may affect the ploidy status in A. atiparanae.

Polyspermy was initiated by microinjecting a multiple number of sperm into the activated and dechorionated eggs of dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). A 10-nl sperm suspension from an albino (recessive trait) male (105, 106, 107 or 108 sperm ml −1) was microinjected into eggs from a wild-type female. Although the rates of embryos developing into the blastula stage in the injection group at the highest sperm concentration were similar to that of the control group, the hatching rates of the injection group were much lower. A large proportion of embryos that developed from the injected eggs was haploid and were mosaics containing haploid cells. Most of the haploid and mosaic embryos inherited only paternally derived alleles in the microsatellite markers (i.e. androgenesis was initiated by injecting multiple sperm). In contrast, some haploid embryos contained both paternal and maternal alleles despite haploidy, suggesting that they were mosaics consisting of cells with either paternal or maternal inheritance. The injected eggs displayed diploid, hypotriploid and triploid cells, all of which included both maternally and paternally derived alleles. One albino tetraploid with only paternal alleles was also observed from the injected eggs. These results suggested that part of the sperm microinjected into the ooplasm should form a male pronucleus/pronuclei, which could develop by androgenesis or could fuse with the female pronucleus/pronuclei. Therefore, microinjection of multiple sperm should be considered a potential technique to induce androgenesis and polyploidy.

The aim of this study was to describe the effect of temperature on the fertilization, early developmental stages, and survival rate of two Neotropical catfishes Pimelodus maculatus and Pseudopimelodus mangurus. After fertilization, the eggs were incubated at 22°C, 26°C, and 30°C, which resulted in fertilization rates of 96.95 ± 1.79%, 98.74 ± 0.76%, and 98.44 ± 0.19% for P. maculatus and 96.10 ± 1.58%, 98.00 ± 0.63%, and 94.60 ± 2.09% for P. mangurus, respectively. For P. maculatus, hatching occurred after 22 h 30 min post-fertilization at 22°C, 16 h 30 min at 26°C, and 11 h 20 min at 30°C, and the hatching rates were 43.87 ± 7,46%, 57.57 ± 17.49%, and 53.63 ± 16.27%, respectively. For P. mangurus, hatching occurred after 28 h 30 min post-fertilization at 22°C and 17 h 30 min at 26°C with respective hatching rates of 45.4 ± 21.02% and 68.1 ± 12.67%. For this species, all embryos incubated at 30°C died before hatching. Additionally, for P. maculatus, the larvae from the lower (22°C) and higher temperatures (30°C) presented increased abnormality rates, as observed in the head, tail and yolk regions. The lowest abnormality rate was detected at 26°C, which was considered the optimal incubation temperature for both species. The developed protocol enables the manipulation of embryonic development, which is important for the application of reproductive biotechniques, including chimerism and chromosome-set manipulation. The data obtained here are also important for the surrogate propagation of this species as P. mangurus was recently categorized as an endangered fish species.

In fish, many factors can affect reproduction during in vitro fertilization, therefore determination of the factors that affect affecting gamete quality is needed. However, few studies have focused on gamete quality and the ploidy status. This study was conducted to elucidate whether oocyte storage can affect ploidy status, survival, and embryo viability in the characid species Astyanax altiparanae. Oocytes were stored in Dulbecco's phosphate-buffered saline (PBS) at 26°C, then aliquots were fertilized immediately after extrusion (control) and also after 60, 120, 180, and 240 min of storage. Fertilization and hatching rates were measured, and the developmental stages were analyzed at each stage before describing the main abnormalities. Ploidy status was analyzed by flow cytometry and blood smear. In the control group, 100% of the samples were diploid. After treatment for 60 min, 95.56 ± 4.44% samples were diploid and 4.44 ± 4.44% were triploid. After 120 min, 94.44 ± 9.62% of the samples was diploid and 5.56 ± 5.56% were triploid; 100% of the samples were diploid after 180 min and, after 240 min, there was no survival. In other treatments, the highest percentage of hatching was after 60 min (88.93 ± 5.15%; P = 0.015), and treatment with 180 min storage resulted in the highest percentage of abnormal larvae (95.76 ± 12.67%; P = 0.012). These results show that oocyte storage can affect ploidy status and may be an interesting parameter for analysis in studies on chromosome set manipulation and micromanipulation.

In fish with external fertilization, sperm must reach the oocyte through the micropyle to enter the cytoplasm. Fertilization success is then influenced by characteristics of oocytes or sperm. In this study, we evaluated oocyte morphology and sperm motility parameters and their effects on the inseminating dose in a teleost fish Astyanax altiparanae. Interestingly, we found one of the lowest yet described inseminating doses in teleosts (2390 spermatozoa oocyte−1 ml−1). Such a fertilization efficacy may be explained by the long duration of sperm motility (>75 s), the small oocyte diameter (695.119 µm), large micropyle diameter (7.57 µm), and the presence of grooves on the oocyte surface that guides spermatozoon to the fertilization area. Additionally, we have described for the first time a structure that combines grooves on the chorion surface and a ridge in the micropylar area.

Primordial germ cell (PGC) transplant is a promising tool in aquaculture; however, successful use of this technique requires in depth knowledge of the early stages of embryo and larval development. The aim of this study was to analyse the effect of different temperatures (22, 26, and 30°C) on the early development of B. amazonicus. The newly fertilized eggs were distributed into tanks with controlled temperature and oxygenation. Samples were collected at pre-established times and analysed under light and fluorescence microscopy. Temperature influenced the speed and duration of each stage of early development, including hatching time. The highest pronuclei fusion rate was observed 8 min post-fertilization (mpf) at 22 and 26°C, and 6 mpf at 30°C. The duration of the 512–1000 blastomeres phase during in the blastocyst stage was 1 h 30 min at 22°C, and 25 min at 26 and 30°C. Hatching occurred at 24 h 30 mpf at 22°C, 16 h post-fertilization (hpf) at 26°C, and 11 h 30 mpf at 30°C. The rate of morphologically normal larvae was 88.34% at 22°C, 90.49% at 26°C, and 73% at 30°C. Malformations of the head, yolk sac, heart, and tail were observed in all temperatures. Nevertheless, B. amazonicus embryos were able to develop satisfactory in all three temperatures tested. These results enable embryo manipulation at different temperatures to optimize the micromanipulation time of embryos and larvae for biotechnological studies.

This study aimed to examine the gonadal morphology of diploid and triploid fish through stereological analysis. Triploid individuals were obtained after temperature shock (40°C for 2 min) at 2 min post-fertilization and reared until 175 days post-fertilization (dpf). Intact eggs were used to obtain the diploids. Gonads were collected for histological analysis at 83, 114, 144 and 175 dpf. Diploid females and males presented normal oogenesis and spermatogenesis through all the experimental period. Conversely, stereological analysis revealed that triploid females were sterile and oogonia were the prevalent cell type in the ovaries. Triploid males presented increased amounts of spermatocyte cysts and a large area of lumen when compared with diploids and in addition the amount of spermatozoa was lower than that observed for diploids. However, some triploid males presented spermatogenesis similar to diploids. Therefore, we concluded that triploidization is an interesting alternative to produce sterile individuals in A. altiparanae.

The aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 µm), midpiece (diameter = 0.75 µm) and a single flagellum (length = 18.67 µm). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22°C, 26°C and 30°C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22°C, 16 hpf at 26°C and 11 hpf at 30°C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22°C, the second polar body was extruded at ≈6 mpf and the male and female pronucleus fused at ≈10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.