Effective UV dose and pressure shock for induction of meiotic gynogenesis in southern flounder (Paralichthys lethostigma) using black sea bass (Centropristis striata) sperm

Female southern flounder (Paralichthys lethostigma) grow 2-3 times larger than males. Therefore, all-female production will maximize profit potential for the culture of this species. We have developed protocols to produce all-female southern flounder through induction of meiotic gynogenesis with heterologous sperm of black sea bass (Centropristis striata). Experiments were conducted to establish these practical methods using a total of 40 spawns from 32 broodstock. The first set of experiments determined the UV dose that genetically inactivated black sea bass sperm, yet retained adequate motility for activation of flounder eggs. Milt from several black sea bass was diluted 1: 10 with Ringer's solution and UV irradiated with doses ranging from 0-130 J/cm(2). Two criteria were utilized to evaluate the UV irradiation effects: percentage of motile sperm and duration of sperm activity. Motility and duration of activity generally decreased with increases in UV dosage. At UV doses greater than or equal to 90 J/cm(2), motility was < 1.5%. Fertilization rates were significantly lower at the highest UV dose of 130 J/cm(2) but were not different for the other treatments. Hatch rate was highest at 70 J/cm(2). A second set of experiments examined appropriate pressure shock protocols for retention of the 2nd polar body in southern flounder eggs after activation with black sea bass sperm. A pressure shock of 8500 psi was initiated at varying times of 1, 2, and 3 min post-fertilization and maintained for 6 min. Eggs that were handled similarly, but not pressure shocked, served as negative controls. Pressure shock applied at either I or 2 min post-fertilization resulted in higher rates of hatch and survival. Using these methods, six separate spawns produced offspring that survived through and beyond metamorphosis. The average fertility ( +/- SEM) was 70.9+12.8%. Of the fertilized eggs, percentage hatch varied with pressure shock initiation times and ranged from 1.48+0.52%(l min)to 0.61+0.11% (3 min). Gynogenetic flounder were sex-reversed to males by high temperature and, upon reaching maturity, expressed motile sperm that resulted in successful fertilization of flounder eggs. These results indicate that the use of UV irradiated sperm from black sea bass for activation of flounder eggs and pressure shock for polar body retention is an effective method to produce gynogenetic offspring. (c) 2006 Elsevier B.V. All rights reserved.