Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish

Abstract

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.

First feeding of diploid and triploid yellowtail tetra Astyanax altiparanae: An initial stage for application in laboratory studies

“…Spawning was induced between males (SL ≈ 6 cm) and females (SL ≈ 9 cm) were using the previous protocol (Yasui et al., ). Briefly, the fish were anesthetized in menthol (100 mg L −1 ), then, injected with a single dose of carp pituitary gland (3 mg kg −1 for males and females), and, afterwards, gametes were obtained 8–10 h later.…”

“…In previous studies from this laboratory, a protocol was developed for in vitro fertilization in laboratory conditions (Yasui et al., ). In vitro fertilization permitted control of the fertilization timing, and, then, experiments involving chromosome and gamete manipulation were developed.…”

Desempenho reprodutivo de lambaris alimentados com grãos secos de destilaria

“…e, em seguida, a papila urogenital de cada um foi seca com toalha de papel e fez-se a compressão abdominal no sentido ântero-posterior. O sêmen foi coletado em Eppendorf de 1000 µL e posteriomente transferido para um tubo de 1,5 mL contendo 400 µL de solução de ringer modificada (128,3 mM NaCl; 23,6 mM KCl; 3,6 mM CaCl2; 2,1 mM MgCl2) para imobilização dos espermatozóides (YASUI et al, 2014). Os Eppendorfs com sêmem foram mantidos em condição de refrigeração (15 0 C).…”

“…Por ser peixe de pequeno porte, o lambari é usado como modelo experimental para outras espécies de grande porte (FELIZARDO et al, 2012;COSTA et al, 2013;YASUI et al, 2014). É utilizado como petisco, isca para a pesca esportiva e extrativa, é comercializado em conserva ou como matéria-prima para o fabrico de farinha de peixe (FERREIRA et al, 2014).…”

Spermatogenesis in the yellowtail tetra Astyanax altiparanae: a histological analyses with emphasis to spermatogonial and spermatid types

“…Popularly known as yellowtail tetra, this is a small fish (reaching a maximum of 20 cm), distributed in High Paraná, Paranapanema, Tibagi and Iguaçu basin (Brazil) in both lotic and lentic environments, being a substantial part of bigger fish diet, besides to be appreciated by human (GARUTTI and BRITISKI, 2000;ORSI et al, 2002;DE BEM, 2009). The choice of species was motivated by its good livestock characteristics, such as ease of handling, small size, early sexual maturity at 4 months of age, and intertidal spawning, which enable it as an appropriate model for investigating the biology of teleost and for the development of distinct studies in laboratory conditions (CASTILHO-ALMEIDA, 2007;YASUI et al, 2014).…”