Reproduction in meagre (Argyrosomus regius) and greater amberjack (Seriola dumerili): a study of environmental and hormonal methods for the control of reproductive maturation and spawning

Abstract

This doctoral thesis aimed to improve current husbandry practices, to enhance reproductive performance under culture conditions, and to support the domestication process of two relevant species for the aquaculture industry: the meagre (Argyrosomus regius) and the greater amberjack (Seriola dumerili).To date, efficient hormonal therapies for spawning induction have enabled overcoming repro-ductive dysfunctions in meagre, achieving consistent egg production and leading to a rapid ex-pansion of meagre commercial production. Nevertheless, their continued necessity underscores the need to refine broodstock rearing protocols. In fact, the lack of ability of hatcheries to obtain spawning without relying on the induction of spawning through exogenous hormones likely reflects improper environmental conditioning of broodstock. Husbandry protocols and hormonal treatments have also been proposed to alleviate repro-ductive dysfunctions in the greater amberjack. Gonadotropin-releasing hormone agonists (GnRHa) has been effective in inducing oocyte maturation (OM) and ovulation, but primarily in breeders housed in sea cages and transferred to land-based facilities during the spawning sea-son, and its effectiveness has been inconsistent in fish reared year-round in land-based tanks with borehole seawater. Moreover, commercial hatcheries mostly rely on wild-caught breeders. Therefore, in addition to the ability to produce high-quality gametes, advancing greater amber-jack aquaculture depends on developing reliable reproduction control strategies for indoor tanks and establishing hatchery-produced populations to facilitate the domestication of this species. To address these challenges, two studies were conducted on meagre, examining the effects of manipulating environmental cues, specifically temperature and salinity, on the gametogenic pro-cess and reproductive success. In parallel, four studies were carried out on greater amberjack. These included the characterization of puberty onset in a first-generation (F1) hatchery-produced population, the development of a hormonal therapy using single-chain recombinant follicle-stimulating (sdrFsh) and luteinizing (sdrLh) hormones to enhance gametogenesis in tank-reared breeders, and the use of the same hormonal tools to induce precocious maturation in pre-pubertal fish. Finally, to support the design of selective breeding programs, the parental con-tribution of broodstock under different reproductive strategies was assessed. In the first experiment conducted on meagre, two broodstocks were maintained in recircu-lating aquaculture systems (RAS) under simulated natural photoperiod and either attenuated sea-sonal water temperature (SeasT, 16.4 to 19.6°C) or relatively constant water temperature (CoT, 19.4 ± 0.6°C). During the spawning season (May), four couples per group were induced to spawn using GnRHa. Gonadal development stage, sperm quality, and plasma sex steroid levels were evaluated prior to hormonal induction, while spawning performance and gamete quality were assessed over the following four weeks. Gametogenesis progressed under both thermal re-gimes; however, the inclusion of winter low temperature in the SeasT group proved beneficial to both sexes. The mean diameter of the largest vitellogenic oocytes prior to GnRHa administration was significantly higher in the SeasT group compared to the CoT group. Female testosterone (T) plasma levels were also significantly higher in the SeasT group, although all other hormone lev-els were similar between treatments. SeasT females exhibited more consistent spawning, higher relative fecundity, and improved 24-hour embryo survival. In males, the negative impact of constant temperature was more pronounced, with CoT males displaying a spermiation index of 0 prior to GnRHa treatment; this was partially reversed by hormonal stimulation over the subse-quent four weeks. Although these findings demonstrate that meagre are capable of undergoing gametogenesis under constant water temperature, an attenuated seasonal thermal regime remains necessary for proper gamete development and effective spawning induction using established GnRHa protocols. In the second experiment on meagre, the effect of salinity variation was tested to examine whether reducing salinity during the reproductive season could stimulate OM, ovulation, and spontaneous spawning in the absence of hormonal treatment, based on the species' euryhaline nature and estuarine spawning behavior. Between May 2019 and May 2020, broodstock were held in RAS at 34‰ salinity before being assigned to four experimental groups on May 2020: CT34 (GnRHa-treated control), SW34 (untreated control), and two low-salinity groups, SW20 and SW12, exposed to 20‰ and 12‰, respectively, and monitored over 25 days. While salinity reduction alone did not trigger spawning, females in SW20 and SW12 showed reduced follicular atresia compared to controls, with SW20 displaying the most advanced oocyte development and highest plasma levels of 17β-estradiol (E2). This suggests that moderate hyposalinity may pro-mote vitellogenesis by reducing osmoregulatory demands, whereas ovarian development was delayed at 12‰, indicating a lower physiological threshold. In males, reduced salinity signifi-cantly improved spermiation, despite similar androgen levels, with the 11-KT/E2 ratio being positively correlated with the spermiation index in the low-salinity groups. Although spawning did not occur without hormonal induction, these results indicate that salinity reduction may help preserve reproductive competence and could serve as a conditioning factor to enhance readiness for spawning induction protocols. In greater amberjack, the first experiment focused on evaluating the onset of puberty in F1 hatchery-produced individuals reared in sea cages over a five-year period. Fish were sampled annually in June, corresponding to the natural peak of the spawning season in the Mediterrane-an. No sexual dimorphism in body weight was observed throughout the study. In females, the ovaries of 1- and 2-year-old fish contained only primary oocytes. By age 3, early vitellogenic oocytes were present, though extensive follicular atresia was evident. At age 4, late vitellogenic oocytes were identified, yet 50% of the females still showed significant atresia. Only at age 5 did ovaries show reduced follicular atresia, indicating further maturation. In contrast, gametogenesis in males was detected as early as age 1, with all 3-year-old males producing collectable viable sperm. Hormonal profiling revealed overall low plasma levels of T, E2, and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) in females, with T and E2 being highest in females at advanced vi-tellogenic. In males, plasma levels of T declined with age, while 11-ketotestosterone (11-KT) and 17,20β-P peaked in 4- and 5-year-old fish, coinciding with improved sperm motility. Im-plants loaded with GnRHa failed to induce spawning in 4-year-old fish and induced only two spawning events in 5-year-old breeders, neither yielding fertilized eggs. These findings suggest that F1 males reach sexual maturity on a timeline similar to that of wild individuals, but exhibit reduced gonad size, while females mature later and also have smaller gonads. The poor response to hormonal induction suggests that Mediterranean hatchery-produced broodstocks exhibit re-productive dysfunctions, warranting further investigation into the effects of age and potential ge-netic limitations of the stock. The second experiment in greater amberjack addressed the reproductive dysfunctions ob-served in broodstock reared in 20-m3 tanks, particularly the inconsistent entry into or comple-tion of vitellogenesis in females, and the low volume and quality of sperm in males. To over-come these limitations, sdrFsh and sdrLh, were administered to 4.8-year-old F1 broodstock. Starting at the onset of gametogenesis (April), both males and females received weekly injections over a period of six weeks, with different combinations and increasing doses of sdrFsh and sdrLh. All treated fish completed gametogenesis, with females progressing to post-vitellogenic oocytes and males producing releasable sperm. Notably, five out of six treated females initiated oocyte maturation prior to GnRHa administration, and at least one spawned spontaneously dur-ing week 4. In contrast, all but one control female failed to complete vitellogenesis and exhibited follicular atresia, rendering them ineligible for spawning induction. Control males produced re-leasable sperm, but with a lower sperm density and significantly lower motility and velocity compared to treated males. Spawning induction with GnRHa in sdrFsh/Lh-treated fish resulted in increased daily relative fecundity and fertilization success, with embryo and larval survival ex-ceeding 50%. These findings demonstrate the efficacy of recombinant gonadotropins in enhanc-ing gametogenesis and inducing spawning in dysfunctional greater amberjack broodstocks reared in tanks, and provide a foundation for future optimization of hormonal protocols.In the third experiment, a combination of sdrFsh and sdrLh was administered to 20-month-old hatchery-produced greater amberjack to test whether the onset of puberty could be advanced in pre-pubertal fish through hormonal stimulation. Using sdrFsh, an initial dose-response trial identified the minimum effective dose to stimulate steroidogenesis in males, as evidenced by GSI values and elevated plasma levels of T and 11-KT levels. In females, the same dose pro-duced only mild increases in plasma levels of T. A 12-week hormonal treatment using weekly sdrFsh/Lh injections commenced in March, coinciding with the expected onset of gametogenesis in the Mediterranean. In treated males, the mean plasma levels of T, 11-KT, and 17,20β-P in-creased significantly compared to controls. By June, complete spermatogenesis and spermiation were achieved, with sperm easily collected from all individuals. The sperm produced by treated males displayed equal or superior concentration and motility compared to naturally matured F1 males reared in sea cages, and outperformed that of wild captive-reared fish from the Mediterra-nean and F1 males from the Atlantic Ocean. In contrast, females failed to respond to the treat-ment; plasma sex steroid levels remained unchanged, and no vitellogenic oocyte development was observed by the end of the trial. These results indicate that shortening the time of puberty to 2 years of age can be achieved in males but not in greater amberjack females. Further research should examine the cause of the unresponsiveness of pre-pubertal females to sdrFsh/Lh, and if it may be possible to stimulate oogenesis in older, pre-pubertal females. In addition to producing high-quality gametes, genetic management is essential for develop-ing hatchery-produced broodstock populations and supporting domestication. To this end, the fourth experiment compared two reproductive strategies, volitional spawning (SPT) and GnRHa-induced spawning (IND) through the administration of slow-releasing implants, in greater am-berjack over a 27-day period, assessing spawning kinetics, egg production, and parentage con-tribution. The SPT group spawned 6 times and the IND group 12 times, with the SPT group showing higher relative fecundity, fertilization success, and embryo survival. However, parent-age analysis revealed a highly skewed female contribution in the SPT group, with 79% of the eggs derived from a single female, while male contributions were more evenly distributed. Alt-hough GnRHa implantation did not improve fertilization parameters, it significantly enhanced the uniformity of female contributions: spawning was synchronized, frequency was reduced, and 95% of total egg production occurred within the first 8 days. Female contribution was equalized (25 ± 1.59%) among individuals, resulting in the successful recovery of 92% of the expected families. These findings demonstrate that GnRHa induction can enhance genetic diversity among offspring, offering a practical tool for improving genetic management and supporting selective breeding programs in greater amberjack hatcheries. Based on the overall findings of this thesis, both meagre and greater amberjack display spe-cies-specific reproductive sensitivities under culture conditions that must be carefully addressed to optimize broodstock management. In meagre, the ability to undergo gametogenesis under con-stant temperature and full-strength seawater confirms the robustness of the species, although at-tenuated seasonal temperature and moderate salinity reduction improve gamete quality and over-all reproductive performance. In contrast, hatchery-produced greater amberjack show to be sus-ceptible to reproductive dysfunctions in a manner similar to what was previously reported for wild-caught individuals, particularly among females. The application of species-specific recom-binant gonadotropins proved effective in enhancing gametogenesis and spawning outcomes in dysfunctional fish maintained in tanks, while also enabling the induction of early puberty in males. Moreover, using GnRHa proved effective in increasing parental contribution, offering a tool for genetic management. Taken together, these results highlight the importance of environ-mental conditioning and tailored endocrine therapies in overcoming reproductive bottlenecks and advancing the domestication process of these two aquaculture species.

Η παρούσα διδακτορική διατριβή είχε ως στόχο τη βελτιστοποίηση των πρακτικών διαχείρισης γεννητόρων και την ενίσχυση της αναπαραγωγικής απόδοσης υπό συνθήκες εκτροφής δύο ειδών ιδιαίτερης σημασίας για τη μεσογειακή υδατοκαλλιέργεια: του κρανιού (Argyrosomus regius) και του μαγιάτικου (Seriola dumerili). Στον κρανιό, η πειραματική διαχείριση περιβαλλοντικών παραμέτρων (θερμοκρασίας και αλατότητας) κατέδειξε ότι η εποχική διακύμανση της θερμοκρασίας και η μέτρια μείωση της αλατότητας βελτιώνουν τη γαμετογένεση, την ποιότητα των γαμετών και την αναπαραγωγική επιτυχία, επιβεβαιώνοντας την ανθεκτικότητα του είδους υπό ελεγχόμενες συνθήκες. Αντίθετα, στους εκτρεφόμενους γεννήτορες μαγιάτικου, παρατηρήθηκαν αναπαραγωγικές δυσλειτουργίες, κυρίως στα θηλυκά άτομα, οι οποίες αντιμετωπίστηκαν επιτυχώς με τη χρήση ανασυνδυασμένων γοναδοτροπινών (sdrFsh, sdrLh). Η εφαρμογή τους προήγαγε τη γαμετογένεση, την ολοκλήρωση της λεκιθογένεσης και την ωοτοκία σε άτομα εκτρεφόμενα σε δεξαμενές, ενώ στα αρσενικά προκάλεσε πρώιμη ήβη και παραγωγή σπέρματος υψηλής ποιότητας. Παράλληλα, η χρήση GnRHa για την επαγωγή της ωοτοκίας εξισορρόπησε τη γονική συμμετοχή, ενισχύοντας τη γενετική ποικιλότητα των απογόνων.