Reproductive biology and endocrinology of the greenback flounder Rhombosolea tapirina (Gunther, 1862)

The greenback flounder Rhombosolea tapirina (Pleuronectidae) is distributed around Southern Australia and New Zealand. There is growing interest in developing an aquaculture industry for greenback flounder in Tasmania and pilot aquaculture schemes are already underway, however, current programs are limited by unreliable egg production. This study provided baseline information on reproductive events under normal reproductive conditions in wild greenback flounder, the stress response of greenback flounder to common husbandry and laboratory aquaculture practices, developed induced ovulation protocols, examined the mechanism of action of hormone treatment and assessed in vitro effectiveness of putative maturational steroids on ovarian tissue. Oocyte size frequency distributions showed that female greenback flounder are multiple ovulators and have group synchronous oocyte development. Plasma and ovarian levels of testosterone (T) and 17˜í‚â§-estradiol (E\\(_2\\)) were elevated in association with vitellogenesis. Plasma levels of 17˜í¬±20˜í‚â§-dihydroxy-4-pregnen-3-one (17,20˜í‚â§P), and 17,20˜í‚â§P-sulphate but not 17,20˜í‚â§P-glucuronide were significantly elevated in ovulated females, whereas ovarian levels of 17,20˜í‚â§P were elevated in association with final oocyte maturation (FOM) and ovulation. Changes in macroscopic testis stage were characterised by few changes in proportions of gamete types between gonad stages, indicating low milt production, even in mature fish. Plasma levels of T were elevated in association with spermatogenesis, but not spermiation. No significant changes in plasma 11-ketotestosterone (11KT), 17,20˜í‚â§P, 17,20˜í‚â§P-sulphate and 17,20˜í‚â§-glucuronide were detected with change in gonad stage of males. The latency of the plasma cortisol response to stress was approximately 10 min. Plasma levels of cortisol were significantly higher in wild fish sampled after capture, confinement and transport, and some routine husbandry practices had the capacity to stimulate elevated cortisol levels for up to 48 h. Hematocrit (Hct) did not change significantly in response to stress, suggesting that either Hct is not effected by stress in this species, or changes in Hct were not detected within the sampling protocol. After exercise, muscle lactate did not significantly change, however, plasma lactate and muscle [H\\(^+\\)] significantly increased and plasma [H\\(^+\\)] significantly decreased, indicating that muscle and blood physiology of greenback flounder do change in response to exercise, but unlike other flatfish, there was little evidence for in situ glycogenesis within white muscle tissue. Female greenback flounder were induced to repeat ovulate using a range of exogenous hormone treatments. Des Gly\\(^{10}\\) [D-Ala\\(^6\\)] LHRH ethylamide (LHRH-a) and human chorionic gonadotropin (hCG), significantly increased the number of ovulations above control levels. Co-administration of LHRH-a and the dopamine antagonist PIM provided no significant advantage over treatment with LHRH-a alone. Dopamine appeared to have an inconsistent effect on reproductive function in greenback flounder. Co-treatment of LHRH-a + T significantly enhanced the ovulatory effects of LHRH-a, suggesting steroid feedback enhances pituitary responsiveness to GnRH. In vitro bioassays indicated that ovarian fragments required pre-treatment with hCG before they were receptive to steroids. In most cases, the maturational response to steroids after priming with hCG exceeded the maturational response to hCG treatment alone. Ovarian fragments were receptive to all steroids at all concentrations tested. There was considerable inconsistency in maturational responses to each steroid and each dose, however, 20˜í¬± and 20˜í‚â§- hydroxylated steroids were most effective at inducing maturation, and 5-pregnene and 5˜í‚â§-pregnane steroids were least effective.