In fish, in vitro fertilization is an important reproductive tool used as first step for application of others biotechniques as chromosome and embryo manipulation. In this study, we aimed to optimize gamete quality and their short-term storage from the yellowtail tetra Astyanax altiparanae, for future application in laboratory studies. Working with sperm, we evaluated the effects of spawning inducers (carp pituitary gland and Ovopel ® [(D-Ala6, Pro9-NEt) -mGnRH + metoclopramide]) and the presence of female on sperm motility. Additionally, we developed new procedures for short-term storage of sperm and oocytes. Briefly, sperm motility was higher when male fish were treated with carp pituitary gland (73.1 ± 4.0%) or Ovopel ® (79.5 ± 5.5%) when compared with the control group treated with 0.9% NaCl (55.6 ± 27.2%; P = 0.1598). Maintenance of male fish with an ovulating female fish also improved sperm motility (74.4 ± 7.4%) when compared with untreated male fish (42.1 ± 26.1%; P = 0.0018). Storage of sperm was optimized in modified Ringer solution, in which the sperm was kept motile for 18 days at 2.5°C. The addition of antibiotics or oxygen decreased sperm motility, but partial change of supernatant and the combination of those conditions improve storage ability of sperm. Fertilization ability of oocytes decreased significantly after storage for 30, 60 90 and 120 min at 5, 10, 15 and 20°C when compared with fresh oocytes (P = 0.0471), but considering only the stored samples, the optimum temperature was 15°C. Those data describe new approaches to improve semen quality and gametes short-term storage in yellowtail tetra A. altiparanae and open new possibilities in vitro fertilization.Keywords: cryopreservation, sperm, oocyte, fish, yellowtail tetra ImplicationsThe yellowtail tetra Astyanax altiparanae is a characin fish largely distributed in Neotropical regions. As a small-sized intertidal spawner, this species is a promising candidate for laboratory and aquaculture technologies including polyploidy, chimerism and nuclear transplantation. However, the first step for such kind of studies is to establish a protocol for in vitro fertilization (IVF) at laboratory conditions. In this work several factors affecting gamete quality were evaluated, and the findings will collaborate for an effective protocol for IVF in this species.

No abstract

Determinação da razão ótima de espermatozóides por ovócitos de piabanha Brycon insignis (pisces -characidae) T1=86,662, T2=173,324, T3=259,986, T4=346,648 and T5=433,310

The production of triploid yellowtail tetra Astyanax altiparanae is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for A. altiparanae species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100) at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5%) and sucrose (74 mM) and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at −20°C) and fixation in saturated NaCl solution, acetic methanol (1:3), ethanol, and formalin at 10% for 30 or 60 days of storage at 25°C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCl2.7H20; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 μg mL−1; preservation procedure: somatic cells (dorsal fin samples) frozen at −20°C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.

ResumoEste trabalho teve como objetivo estudar a biologia reprodutiva da tabarana Salminus hilarii, na represa de Três Marias (MG). Foram determinados, a partir de exemplares capturados durante 20 meses, os seguintes parâmetros: comprimento padrão (CP), peso corporal (PC), índices gonadossomático (IGS), hepatossomático (IHS), de gordura abdominal (IGA) e estágio de maturação (em repouso, em maturação, maduro ou esgotado). Verificou-se que o período de reprodução da tabarana se concentra nos meses finais e iniciais do ano, podendo ser classificada como de desova total. Foi avaliado, ainda, o grau de aspereza ao tato das espículas na nadadeira anal dos machos, sendo observados incrementos crescentes conforme avançava o grau de maturação dos exemplares.Palavras-chave: reprodução, Salminus, reservatórios, característica sexual secundária, tabarana. AbstractReproductive biology of Salminus hilarii (osteichthyes, characidae) from a brazilian man-made lake This work was carried out at Três Marias dam, Minas Gerais State, Brazil, with the aim of studying the male and female reproductive biology of the fish Salminus hilarii in a man-made lake. From fishes captured during the period April/1982 to november/1984 the following results were obtained: the body length, body weight and the indices: gonadossomatic, hepatossomatic and abdominal fat; stages of ovarian development (dormant, in maturation, ripe and spawned or exhausted). It was verified that the spawning period S. hilarii occurred at the end and at the beginning of each year of this study and this fish species could be classified as a total spawner. From the observations of the level of the toughness of the anal fin of male fish (sexual secondary characteristic) it was verified that roughness was more evident in fish with ovaries in advanced maturation stage.