Spawning induction of first-generation (F1) greater amberjack Seriola dumerili in the Canary Islands, Spain using GnRHa delivery systems

The development of a sustainable aquaculture industry requires the closing of the life-cycle of a potential species in captivity, and the establishment of breeding selection programs using hatchery-produced breeding stocks. The greater amberjack Seriola dumerili is a cosmopolitan pelagic species that has been considered as a good candidate for the species diversification of aquaculture production in the Mediterranean region. However, commercial production is still very limited due to bottlenecks in reproduction, larval rearing and management control during grow out. The aim of the present study was to examine the reproductive development of hatchery-produced greater amberjack and to develop a spawning induction protocol based on the use of gonadotropin releasing hormone agonist (GnRHa) in a controlled-release delivery system. The results showed that hatchery-produced greater amberjack undergo normal gametogenesis and can be induced to undergo maturation, ovulation and spawning after multiple administrations of GnRHa implants, over an extended spawning period lasting from May to September in the Canary Islands, Spain. The use of GnRHa-delivery systems resulted in multiple spawns of fertilized and viable eggs. Egg production was high and egg quality adequate for the implementation of larval rearing for commercial purposes. The handling required to administer the GnRHa implants during the prolonged spawning season did not result in any negative effect on the welfare and reproductive performance of the fish based on evaluation of several biochemical parameters. The developed reproduction control method shows great potential to advance the commercial production of greater amberjack, by enabling the use of hatchery-produced broodstocks for further breeding selection.Postprin

Endocrine regulation of long-term enhancement of spermiation in meagre (Argyrosomus regius) with GnRHa controlled-delivery systems.

Abstract Meagre (Argyrosomus regius) undergo spermatogenesis and spermiation when reared in captivity, but often produce low milt volumes, sometimes with reduced quality and for a limited time period. In the present study we a) compared the efficiency of gonadotropin releasing hormone agonist (GnRHa) implants versus injections on testicular stimulation and spermiation enhancement, b) investigated the effect of GnRHa on the endocrine spermiation regulation (sex steroid hormones), and c) evaluated a commercial induced spawning simulation scenario. Firstly, males (n = 5) were injected with 15.0 ± 0.2 μg GnRHa kg−1 (Injections) or implanted with 51.0 ± 5.1 μg GnRHa kg−1 (Implant) and compared their sperm production response. Secondly, the best hormonal treatment (Implant) was tested treating males (n = 8) with 57.5 ± 7.5 μg GnRHa kg−1 every 3 weeks for a period of 70 days. Milt production was improved by the GnRHa implants with only minor sperm quality alterations (improved sperm motility percentage). Elevated plasma testosterone (T) and 11-ketotestosterone (11-KT) levels were recorded in response to GnRHa implants, while no significant difference for 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was observed. In the commercial induced spawning simulation, it was shown that meagre females are capable of on-demand induction of spawning at random intervals (5–21 days) using GnRHa injections, over a period of at least 2.5 months. During this period, spermiation enhancement was achieved with GnRHa implants every 3 weeks, producing sperm with stable, in general, quality and motility parameters. Percentage of motile cells, motility duration and density fluctuated significantly, but remained within levels that are considered appropriate for high fertilization success in this species

Spawning kinetics and parentage contribution of European sea bass (Dicentrarchus labrax) broodstocks, and influence of GnRHa-induced spawning

Abstract Increasing parentage contribution in aquaculture broodstocks is important, in order to take full advantage of the available genetic makeup of the chosen fish, and to avoid inbreeding and loss of allele diversity over subsequent production generations. European sea bass (Dicentrarchus labrax) broodstocks were evaluated over two reproductive seasons to examine spawning kinetics, egg production, and parentage contribution during spontaneous/volitional spawning. In addition, we obtained preliminary results on the potential of a hormonal therapy to synchronize spawning and increase parentage contribution. Spawning lasted between 25 and 66 days in January-March and consisted of 12–21 daily spawns per broodstock, with individual females spawning 1–5 times and males participating in 1–8 spawns during each reproductive season. Daily fecundity was variable during the season, without any trend, and so were all the examined egg/larval quality parameters. Parentage assignment of the produced families indicated that the majority of progeny from a whole season may belong to a very small number of breeders, with four females producing up to 80 % of the analyzed eggs, while a single male may sire up to 57 % of the fertilized eggs. No significant improvement in the overall parentage contribution was obtained with the hormonal treatment, using gonadotropin releasing hormone agonist (GnRHa). Nevertheless, the daily fecundity was higher, and parentage of the eggs from the first spawn after GnRHa treatment was more equally distributed to multiple males/females, compared to any volitional spawns. The study demonstrates the need to further improve parentage contribution in European sea bass aquaculture, through synchronization of maturation and spawning. Although the GnRHa induction experiment was not replicated in the present preliminary study, the results suggest that hormonally-induced synchronization of maturation may have the potential of producing a larger number of progenies from more families, from where to select the next generation of breeders for a breeding program

The observed oogenesis impairment in greater amberjack Seriola dumerili (Risso, 1810) reared in captivity is not related to an insufficient liver transcription or oocyte uptake of vitellogenin

The greater amberjack Seriola dumerili is an excellent candidate for the Mediterranean aquaculture, due to its large body size and high growth rate, as well as its high flesh quality and commercial value worldwide. For its successful incorporation in the aquaculture industry, an in-depth understanding of the reproductive function of the species under rearing conditions is necessary, since completion of oogenesis in captivity is currently a bottleneck for the commercial production of the species. Liver and ovary samples from wild and captive-reared greater amberjack females were collected at three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). The cDNAs of three vitellogenins (VtgA, VtgB and VtgC) were partially sequenced and a qRT-PCR for their expression was used to compare ovarian maturity stage and liver vitellogenin transcript levels between wild and captive-reared individuals. An extensive atresia of late vitellogenic follicles, which prevented any further oocyte development and spawning was observed in captive-reared individuals during the ADVANCED phase. The expression levels of the three vitellogenins, as well as the amount of yolk globules in vitellogenic oocytes, did not differ significantly between captive-reared and wild females, indicating that the observed oogenesis impairment in greater amberjack reared in captivity was not related to an insufficient liver synthesis or a reduced oocyte uptake of vitellogenin

Description of the annual reproductive cycle of wreckfish; Polyprion americanus, in captivity

Successful spontaneous reproduction and the production of viable offspring of wild fish in captivity can take some years of adaptation, and may also involve different environmental conditions, sex ratios and densities compared to natural populations. We followed the reproductive cycle of wreckfish Polyprion americanus—a late maturing, deep-sea benthic species—in captivity in three different broodstocks in Spain under natural photoperiod and temperature, and one broodstock in Greece under a constant temperature of 16 °C for two consecutive years, to describe the reproductive cycle of the species and the associated sex steroid hormone profiles. Oogenesis begun in the fall and post-vitellogenic oocytes of 1250 μm were present between March and June. Males were in spermiation condition and produced good-quality sperm throughout the year, regardless of the temperature profile to which they were exposed. Some females completed oogenesis, underwent oocyte maturation, and spawned spontaneously under both constant and fluctuating temperatures. The sex steroid hormones of both males and females followed the already-known profiles during fish gametogenesis, except for 17,20β-dihydroxy-progesterone, which did not seem to be related to either female or male maturation. The female reproductive dysfunctions that were identified included (a) the arrest of oogenesis at the cortical alveoli stage in certain females; (b) the failure to undergo oocyte maturation in others; and (c) the production of large percentages of unfertilized eggs from the females that spawned spontaneously. Our study suggests that reproduction in captivity is possible in wreckfish maintained under typical marine aquaculture conditions, but that reliable spawning and production of high-quality eggs may require some years of adaptation to captivity, before the reproductive dysfunctions will be overcome.Versión del edito

Wreckfish (Polyprion americanus). New Knowledge about reproduction, larval husbandry and nutrition. Promise a new species for aquaculture

Four different wreckfish (Polyprion americanus) broodstock batches were maintained in research facilities under different photo and thermo-period conditions, one in Greece, the Helenic Center for Marine Research (HCMR, n = 3) and three in Spain: Instituto Español de Oceanografía (IEO, n = 13) in Vigo, Aquarium Finisterrae (MC2, n = 21) in A Coruña and Consellería do Mar (CMRM, n = 11). The CMRM includes two centers that work together: Instituto Galego de Formación en Acuicultura (IGAFA) and Centro de Investigacións Mariñas (CIMA), both in Pontevedra. During the five years of the project DIVERSIFY (Exploring the biological and socio-economic potential of new-emerging candidate fish species for the expansion of the European aquaculture industry, 2013–2018) works focused on the reproductive biology of the species, broodstock, and larvae nutrition and development of incubation and larval rearing protocols have been carried out. In terms of reproduction, catch methods of new wild animals, the reproductive cycle, sperm characteristics evaluation, and spontaneous and induced spawning methods have been described for wreckfish. Regarding nutrition, the positive effect of two types of enrichment on the fatty acid profiles of Artemia and rotifer has been verified. The relationship between the fatty acid profile of the diets supplied to the broodstock and the fatty acid profile obtained in the oocytes and eggs of the females fed with different diets, has also been demonstrated. Finally, early larval ontogeny has been described and incubation and larval rearing protocols have been proposed based on the results obtained in the different experiments of temperature, growth, survival, and larval feeding that were carried out.Versión del edito

Αναπαραγωγική βιολογία του μαγιάτικου Seriola dumerili σε συνθήκες ιχθυοκαλλιέργειας

Members of the genus Seriola have been notable species for aquaculture production worldwide, with the greater amberjack being one of the most prominent species, due to its cosmopolitan distribution and market acceptability, high growth rates and large size. However, the prerequisite to sustainable aquaculture production -i.e. controlling reproduction in captivity- has been lacking for greater amberjack. The objective of the present doctoral thesis was to describe the reproductive function of wild and captive reared fish, examine the reproductive maturation potential of fish reared in land-based tanks and sea cages and develop broodstock management methods and spawning induction protocols using gonadotropin releasing hormone agonists (GnRHa). The effectiveness of GnRHa injections versus GnRHa implants of sustained release was examined, and the best method was then optimized by examining (a) optimal dose and (b) time of application. The extent of spermiation period and sperm quality parameters of breeders maintained in different locations and facilities was examined using computer assisted sperm analysis (CASA) to examine any variations over time and after spermiation enhancement trials. Finally, the spawning induction method was also evaluated for the first time on first generation (F1) breeders in Spain to ensure uninterrupted production of fingerlings for genetic breeding programs.A comparative study of wild and captive greater amberjacks was carried out to compare the endocrinological status of the pituitary-gonad axis related with reproductive developmental stage. A total of 33 (14 males and 19 females) wild and 24 (12 males and 12 females) captive-reared greater amberjack breeders were sampled at three different phases of the reproductive cycle. The sampled wild females were separated in two categories according to the histological analysis of their ovaries, in vitellogenic in early May and spawning capable in late May-late June. Between these two categories a 3 to 4-fold increase of the gonadosomatic index (GSI) was recorded. At the same time a corresponding increase was recorded for pituitary follicle stimulating hormone (FSH) content and plasma testosterone (T), while almost a 10-fold increase was observed in pituitary luteinizing hormone (LH) content. An increasing trend of plasma 17β-estradiol (E2) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was also recorded between the two reproductive stages. On the other hand, sampled captive females were separated into four categories of reproductive development, since extended follicular atresia (AT) was observed from late May, with the majority of the fish that had already ceased their reproductive development showing signs of regressed or regenerating ovaries, possibly due to repeated handling. Pituitary content of FSH and LH, fshβ and lhβ expression and plasma levels of FSH and LH remained unchanged, in contrast with the plasma E2 and T that were reduced towards the regenerating phase, and 17,20β-P which showed the opposite pattern. No significant hormonal differences, except in 17,20β-P, were recorded between captive and wild vitellogenic females, in contrast to spawning capable females where pituitary LH content, plasma FSH and T were found to be different. Overall, the captive females lagged behind compared to the wilds on reproductive development and this was probably related with the repetitive handling in sea cages.Wild male fish were found to be in corresponding reproductive stages with females, as expected. Pituitary LH content, plasma T, 17,20β-P and GSI of wild males exhibited a 3 to 4-fold increase, while an increasing trend of pituitary FSH content, lhβ expression levels and plasma 11-ketotestosterone (11-KT) was also observed between the developing and spawning capable reproductive stages, while an opposite trend was observed in plasma LH. On the other hand, sampled captive males fell into three categories of reproductive development, showing significant differences from their wild counterparts even from the developing phase in four out of the ten measured parameters. During spawning capable phase, the situation was even worst, since in almost all the measured parameters reduced levels between wild and captive males were recorded, being statistically significant or showing a strong downward trend. Therefore, the captive males apart from the handling stress that could be related with the worst reproductive development compared to the wilds after the 2nd sampling onwards, showed differences from the first sampling, a more pronounced effect of the captivity compared to the females.To develop broodstock management and spawning induction methods for greater amberjack, it was examined the possibility of rearing fish either in tanks or sea cages, and evaluated their spawning performance. The advantages of sea cage rearing and spawning include optimal environmental conditions and welfare, and low cost of fish maintenance. The advantages of tank rearing and spawning include biosecurity, ease of management and handling operations, and control of egg collection. Females reared in tanks supplied with well seawater throughout the year exhibited a significant reproductive dysfunction, such as limited gametogenesis, lower oocyte diameter at the peak of the reproductive season, low female eligibility for spawning induction and almost 0% fertilization success. On the contrary, females in three different broodstocks reared in sea cages for three consecutive years underwent vitellogenesis completely, and almost all females were eligible for spawning induction. Males reared under both conditions exhibited reduced sperm production compared to the wild, but unlike the situation in the females, tank rearing did not seem to have any negative effects on sperm quality parameters compared to rearing in sea cages. Spawning performance (eggs collected and fertilization) was best when fish were reared in sea cages during the year and then moved to land-based tanks for spawning after GnRHa treatment, resulting in the production of large numbers of eggs of adequate quality for commercial larval rearing of the species.The present study examined also two methods for the induction of oocyte maturation/ovulation and spawning, which are based on the induction of endogenous LH release from the pituitary, through the use of GnRHa either in the form of sustained-release delivery systems (implants) or injections (acute release). The stock (n = 28) consisted of wild fish captured in 2011 and each GnRHa administration method was conducted in two replicates with 6 and 8 fish per tank, at a 1:1 sex ratio. Fish were given a GnRHa injection once a week (three administrations), or a GnRHa implant every 2 weeks (two administrations). Mean daily relative fecundity was significantly higher in the implanted fish compared to the injected fish. Total relative fecundity was also significantly higher in the implanted fish compared to the injected ones, but there were no differences in the quality of eggs in terms of fertilization, 24-h embryo survival, hatching and 5-d larval survival. The number of females with fully vitellogenic oocytes eligible for induction of spawning decreased from the initial to the final sampling, from 7 to 6 females for the GnRHa implant treatment and from 7 to 3 females for the GnRHa injection treatment. The use of GnRHa implants apparently promoted the proper endocrine changes leading to multiple cycles of oocyte maturation, ovulation and spawning, and thus producing larger numbers of eggs. In addition, the use of GnRHa implants may be more appropriate in greater amberjack than multiple injections because (a) it also stimulates vitellogenesis and (b) involves less handling of the fish.After the successful control of oocyte maturation, ovulation and spawning using hormonal therapies in the form of controlled-release delivery systems of GnRHa, the objective was to optimize the protocol and (a) determine the most efficient GnRHa implant dose and (b) examine the extent of the spawning induction period in greater amberjack reared in sea cages throughout the year, based on the resulting spawning kinetics, egg production and quality. Wild-caught greater amberjack were maintained in two commercial facilities, and were transferred to onshore tanks for spawning after treatment with GnRHa implants (a) of different doses (25 and 75 μg kg-1 body weight), or (b) at different times during the expected spawning season (June-July). Both doses were equally effective, and two consecutive implantations in 2 weeks resulted in mean (±SD) total relative fecundity of 185,221± 91,311 to 199,492 ± 16,973 eggs kg -1 in 11-18 spawns, without any significant differences in egg quality. Another noteworthy observation from the present study was that “spent” females or with smaller remaining vitellogenic (Vg) oocytes after spawning induction in the onshore tanks, recovered to their initial reproductive stage –in terms of maximum Vg oocyte diameter- after a “resting” period of 14-28 days in sea cages, while their repetitive handling at the time of reproductive stage evaluation didn’t caused any negative effects on their reproductive development. Therefore, it was shown that a protocol where breeders are maintained in sea cages, are taken at different times and are induced to spawn with a GnRHa implant of a recommended dose of 50 μg kg-1, can be used successfully during a period of at least 2 months when seawater temperature is 19-24°C.In many fish species, the F1 fish face reproductive dysfunctions in captive conditions, such as lower reproductive success, production of unfertilized eggs and lack of spawning, compared to the wild-caught breeding animals. To examine this possibility in greater amberjack, hatchery-produced fish were examined during the expected spawning season and were hormonally induced to spawn. The results showed that hatchery-produced greater amberjack undergo normal gametogenesis and can be induced to undergo maturation, ovulation and spawning after multiple administrations of GnRHa implants, over an extended spawning period lasting from May to September in the Canary Islands, Spain. The use of GnRHa-delivery systems resulted in multiple spawns of fertilized and viable eggs. The developed reproduction control method shows great potential to advance the commercial production of greater amberjack, by enabling the use of hatchery-produced broodstocks for further breeding selection. However, more research is needed to provide solutions for selective breeding in greater amberjack, due to impossible use of the in vitro fertilization protocol in this species.Finally, sperm quality was evaluated using CASA before and after GnRHa administration and the extent of the spermiation period was determined. Males were in spermiation throughout the monitoring period from the 30th of May until the 18th of July, at temperatures between 19-24ºC. However, lower sperm motility duration, density and survival under cold storage were observed from early July onwards. Sperm quality did not recover after the end of spawning induction experiment in tanks and the return of the fish for 14-28 days to the sea cage in mid-July, which could be related to the high temperatures of this period. An improvement trend was observed in the quality of the milt collected on day 7 after a single GnRHa administration, but a significant decrease was observed on day 21 in sperm density, survival under cold storage and straight line velocity (VSL). On the contrary, a double GnRHa administration spaced 14 days apart maintained the same sperm quality for a longer period of 29 days. Further experimentation needs to be done in greater amberjack to ensure adequate sperm production for high fertilization success.Considering all the above, the captive greater amberjack is sensitive to handling at the time before the onset of spawning period regarding normal reproductive development. In males, there is a clear effect of confinement on reproductive development. In contrast, when the fish are left unhandled before the spawning period the repetitive handling do not cause any negative effect on their reproductive development. Interestingly, the females recover shortly to their initial reproductive stage after spawning induction after a resting period in sea cages, opposite to the males. A protocol where fish are maintained in sea cages throughout the year, and transferred to onshore tanks after spawning induction with GnRHa implants at a dose of 50 μg GnRHa kg-1 body weight (BW) seems to be best in the Mediterranean. Both females and males, at temperatures ranging from 19-24oC in June-July, can be used to ensure adequate egg production.Το μαγιάτικο είναι ένα από τα υποψήφια είδη για τη διαφοροποίηση της παραγωγής της ιχθυοκαλλιέργειας λόγω των συγκριτικών πλεονεκτημάτων του σε σχέση με άλλα είδη, όπως η ευρεία κατανομή του στις εύκρατες περιοχές, ο γρήγορος ρυθμός ανάπτυξης και το μεγάλο μέγεθός του, κατάλληλο για την παραγωγή διαφορετικών προϊόντων από το εδώδιμο τμήμα του. Ωστόσο, δεν έχει επιτευχθεί ακόμη ο έλεγχος της αναπαραγωγής του σε συνθήκες εκτροφής, το οποίο δημιουργεί προβλήματα στην απρόσκοπτη εμπορική παραγωγή του. Αυτό οφείλεται σε αναπαραγωγικές δυσλειτουργίες που παρατηρούνται κατά την διάρκεια της εκτροφής, όπως συμβαίνει και με άλλα νέο-εισερχόμενα είδη. Στόχος της παρούσας έρευνας ήταν να περιγραφεί η αναπαραγωγική λειτουργία εκτρεφόμενων και άγριων ψαριών σε διαφορετικά επίπεδα του άξονα υποθαλάμου – υπόφυσης – γονάδων. Επίσης, να ελεγχθούν οι χερσαίες δεξαμενές και οι ιχθυοκλωβοί ως συστήματα διαχείρισης γεννητόρων για την βέλτιστη γαμετογένεση του μαγιάτικου σε συνθήκες εκτροφής και να γίνει σύγκριση του αναπαραγωγικού δυναμικού των γεννητόρων στις διαφορετικές συνθήκες. Αναπτύχθηκαν μέθοδοι διαχείρισης γεννητόρων, πρωτόκολλα πρόκλησης ωοτοκίας με χρήση αγωνιστών της εκλυτικής ορμόνης των γοναδοτροπινών (GnRHa) και συλλογής αυγών στις υπάρχουσες εγκαταστάσεις της μεσογειακής ιχθυοκαλλιέργειας, με σκοπό να υπερκεραστούν οι αναπαραγωγικές δυσλειτουργίες που εντοπίζονται στο είδος. Σε επόμενο στάδιο, έγινε σύγκριση διαφορετικών μεθόδων χορήγησης GnRHa (ενέσεις για την άμεση εισαγωγή της δραστικής ουσίας στο κυκλοφορικό σύστημα του ψαριού, και εμφυτεύματα για την ελεγχόμενη έκλυση της δραστικής ουσίας, αντίστοιχα) όσον αφορά την ποσότητα και ποιότητα των παραγόμενων αυγών, αλλά και της αναπαραγωγικής κατάστασης των θηλυκών. Στη συνέχεια, η καλύτερη αυτών των μεθόδων ελέγχθηκε για την εξακρίβωση της βέλτιστης δόσης GnRHa, μέσω της αξιολόγησης των ίδιων παραμέτρων, ενώ παράλληλα ελέγχθηκε η βέλτιστη περίοδος εφαρμογής στην εκτιμώμενη περίοδο ωοτοκίας στην Μεσόγειο. Όσον αφορά τους αρσενικούς γεννήτορες, αξιολογήθηκε η παραγωγή και ποιότητα σπέρματος σε διαφορετικές περιόδους κατά την περίοδο σπερμίασης, αλλά και μετά από μονή ή διπλή χορήγηση εμφυτευμάτων ελεγχόμενης έκλυσης GnRHa, με τη χρήση «αυτοματοποιημένης ανάλυσης ποιότητας σπέρματος» (CASA). Τέλος, ελέγχθηκε η αναπαραγωγική λειτουργία μετά από πρόκληση ωοτοκίας σε εκτρεφόμενους γεννήτορες πρώτης γενιάς (F1), ώστε να αποκλειστούν προβλήματα που παρατηρούνται σε νεο-εισερχόμενα είδη στην διαδικασία της εκτροφής, τα οποία περιορίζουν την απρόσκοπτη εφαρμογή προγραμμάτων επιλογής γεννητόρων.Για να ελεγχθεί η αναπαραγωγική κατάσταση εκτρεφόμενων ψαριών έγινε συγκριτική μελέτη με αλιευμένα ψάρια σε διαφορετικά επίπεδα του αναπαραγωγικού άξονα υποθαλάμου-υπόφυσης-γονάδων. Συνολικά σε 33 αλιευμένα ψάρια (14 αρσενικά και 19 θηλυκά) και 24 εκτρεφόμενα (12 αρσενικά και 12 θηλυκά) έγιναν δειγματοληψίες σε τρεις διαφορετικές φάσεις του αναπαραγωγικού κύκλου. Στα αλιευμένα θηλυκά πραγματοποιήθηκε ιστολογική επεξεργασία των ωοθηκών τους, σύμφωνα με την οποία κατατάχθηκαν σε δύο κατηγορίες ανάλογα με την αναπαραγωγική τους κατάσταση. Συγκεκριμένα, διαχωρίστηκαν σε αυτά που ήταν σε φάση ανάπτυξης των ωοκυττάρων τους, όταν η δειγματοληψία έγινε αρχές Μαΐου και σε αυτά που ήταν σε φάση ωοτοκίας όταν η δειγματοληψία έγινε τέλη Μαΐου – τέλη Ιουνίου. Μεταξύ των ψαριών των δύο κατηγοριών υπήρξε σημαντική αύξηση του γοναδοσωματικού δείκτη (GSI) (3-4 φορές). Παρόμοια αύξηση παρατηρήθηκε στην συγκέντρωση της ωοθυλακιοτρόπου ορμόνης (FSH) στην υπόφυση και στα επίπεδα της τεστοστερόνης (Τ) στον ορό του αίματος, ενώ παρατηρήθηκε σημαντική αύξηση (περίπου 10 φορές) της συγκέντρωσης της ωχρινοτρόπου ορμόνης (LH) στην υπόφυση. Καταγράφηκε αυξανόμενη τάση, αλλά όχι στατιστικά σημαντική, της συγκέντρωσης της 17β-οιστραδιόλης (E2) και της προγεστερόνης 17α,20β-διυδροξυ-4-πρεγνεν-3-όνη (17,20β-P) στον ορό του αίματος μεταξύ των δύο αναπαραγωγικών φάσεων (φάση ανάπτυξης και ωοτοκίας – developing, spawning capable). Η έκφραση των fshβ και lhβ, αλλά και τα επίπεδα της FSH και LH στον ορό του αίματος δεν διαφοροποιήθηκαν σημαντικά. Από την άλλη, τα εκτρεφόμενα θηλυκά κατατάχθηκαν σε τέσσερις κατηγορίες αναπαραγωγικής ανάπτυξης (developing, spawning capable, regressing, regenerating), δεδομένου ότι παρατηρήθηκε εκτεταμένη εκφύλιση των ωοθυλακίων μετά το τέλος Μαΐου, όπου η πλειοψηφία των ψαριών είχε ολοκληρώσει τον αναπαραγωγικό τους κύκλο, πιθανότατα λόγω των επαναλαμβανόμενων χειρισμών. Η συγκέντρωση της FSH και της LH στην υπόφυση, η έκφραση των fshβ και lhβ και τα επίπεδα της FSH και LH στον ορό του αίματος παρέμειναν στα ίδια επίπεδα, σε αντίθεση με τα επίπεδα της E2 και T στον ορό του αίματος, τα οποία μειώθηκαν, ενώ η 17,20β-P έδειξε αντίθετη φορά. Δεν παρατηρήθηκαν σημαντικές διαφορές μεταξύ των αλιευμένων και εκτρεφόμενων ψαριών στις μετρούμενες παραμέτρους, εκτός από την 17,20β-P, κατά τη φάση ανάπτυξης των ωοκυττάρων. Αντίθετα, στη φάση ωοτοκίας η συγκέντρωση της LH στην υπόφυση, αλλά και της FSH και της Τ στον ορό του αίματος διέφεραν στατιστικά σημαντικά μεταξύ των δύο πληθυσμών. Συνολικά, τα εκτρεφόμενα θηλυκά υστερούσαν σε αναπαραγωγική ανάπτυξη σε σχέση με τα αλιευμένα, γεγονός που πιθανότατα σχετίζεται με τους επαναλαμβανόμενους χειρισμούς στους ιχθυοκλωβούς.Όσον αφορά τα αλιευμένα αρσενικά, βρέθηκαν σε αντίστοιχες αναπαραγωγικές φάσεις με τα θηλυκά, όπως ήταν αναμενόμενο. Η συγκέντρωση της LH στην υπόφυση, της Τ και 17,20β-P στον ορό του αίματος και ο GSI αυξήθηκαν σημαντικά (3-4 φορές), ενώ παρατηρήθηκε τάση αύξησης στην συγκέντρωση της FSH στην υπόφυση, την έκφραση του lhβ και τη συγκέντρωση της 11-κετο-τεστοστερόνης (11-ΚΤ) στον ορό του αίματος μεταξύ των φάσεων της ανάπτυξης (developing) των όρχεων (σπερματογένεση) και σπερμιογένεσης-σπερμίασης (spawning capable), ενώ αντίθετη τάση παρατηρήθηκε στην LH του ορού του αίματος. Δεν παρατηρήθηκε διαφορά μεταξύ των δύο αναπαραγωγικών φάσεων στην έκφραση του fshβ και της FSH στον ορό του αίματος. Από την άλλη, τα εκτρεφόμενα αρσενικά χωρίστηκαν σε τρεις κατηγορίες αναπαραγωγικής ανάπτυξης – developing, spawning capable, regressing - σύμφωνα με την ιστολογική αξιολόγηση των όρχεων, έχοντας σημαντικές διαφορές από τα αντίστοιχα αλιευμένα αρσενικά, ακόμη και από την φάση ανάπτυξης των όρχεων σε πέντε (fshβ, FSH και LH στον ορό του αίματος, 17,20β-P και GSI) από τις δέκα μετρούμενες παραμέτρους. Κατά την φάση σπερμιογένεσης - σπερμίασης, η κατάσταση έγινε ακόμη χειρότερη στη σύγκριση με τα αλιευμένα αρσενικά, αφού σχεδόν σε όλες τις παραμέτρους παρατηρήθηκαν μειωμένες τιμές των εκτρεφόμενων αρσενικών, είτε στατιστικά σημαντικές ή δείχνοντας τάση μείωσης. Επομένως, τα εκτρεφόμενα αρσενικά εκτός από την καταπόνηση από τους επαναλαμβανόμενους χειρισμούς στους ιχθυοκλωβούς από τη 2η δειγματοληψία και μετά, που πιθανότατα επηρέασαν αρνητικά την αναπαραγωγική τους ανάπτυξη σε σχέση με τα αλιευμένα, είχαν διαφορές ακόμη και από την 1η δειγματοληψία, δηλαδή η καταπόνηση λόγω αιχμαλωσίας φαίνεται να είναι σημαντικότερη από ότι στα θηλυκά.Ένας επιπλέον στόχος αυτής της μελέτης ήταν η ανάπτυξη μεθόδων διαχείρισης γεννητόρων, αξιολόγησης της αναπαραγωγικής κατάστασης και πρόκλησης ωοτοκίας σε μαγιάτικα που διατηρούνται τόσο σε δεξαμενές, όσο και σε ιχθυοκλωβούς. Τα πλεονεκτήματα της εκτροφής σε ιχθυοκλωβούς είναι οι βέλτιστες περιβαλλοντικές συνθήκες της θάλασσας, οι συνθήκες ευημερίας των ψαριών και το χαμηλό οικονομικό κόστος διατήρησης των γεννητόρων, ενώ στα πλεονεκτήματα της εκτροφής σε δεξαμενές περιλαμβάνονται η βιοασφάλεια, η ευκολία στη διαχείριση των γεννητόρων και ο έλεγχος στη συλλογή των αυγών. Ωστόσο, στα θηλυκά που παρέμειναν σε δεξαμενές για όλη τη διάρκεια του χρόνου και τους παρεχόταν θαλασσινό νερό γεώτρησης παρατηρήθηκαν αναπαραγωγικές δυσλειτουργίες, όπως η ελλιπής γαμετογένεση, μικρότερη διάμετρος των ωοκυττάρων στα μέσα της αναπαραγωγικής περιόδου, και σχεδόν 0% ποσοστά γονιμοποίησης των αυγών που παρήγαγαν. Αντίθετα, τρεις διαφορετικές ομάδες γεννητόρων που εκτράφηκαν σε ιχθυοκλωβούς για τρία χρόνια ολοκλήρωσαν τη λεκιθογένεση και, σχεδόν, όλα τα θηλυκά ήταν σε κατάλληλη αναπαραγωγική κατάσταση για την επαγωγή της ωρίμανσης, ωορρηξίας και ωοτοκίας. Από την άλλη, τα αρσενικά τόσο στις δεξαμενές όσο και στους ιχθυοκλωβούς είχαν μειωμένη παραγωγή σπέρματος σε σχέση με άγρια ψάρια στη φύση, αν και το περιβάλλον των δεξαμενών δεν φάνηκε να επηρεάζει τα αρσενικά τόσο όσο τα θηλυκά, σε σχέση με τα ψάρια των ιχθυοκλωβών. Ο καλύτερος τρόπος, όσον αφορά τη συλλογή αυγών και ποσοστό γονιμοποίησης αυτών, φάνηκε να είναι ο συνδυασμός των δύο συστημάτων, δηλαδή όταν τα ψάρια διατηρούνταν στους ιχθυοκλωβούς για όλο το χρόνο και μεταφέρονταν στις χερσαίες δεξαμενές μετά από θεραπεία με GnRHa, όπου και παρήγαγαν μεγάλες ποσότητες αυγών καλής ποιότητας.Η μελέτη εξέτασε επίσης δύο μεθόδους πρόκλησης ωρίμανσης των ωοκυττάρων, ωορρηξίας και ωοτοκίας, οι οποίες βασίζονται στην απελευθέρωση της ενδογενώς παραγόμενης LH, μέσω της χορήγησης GnRHa με μορφή εμφυτευμάτων ελεγχόμενης έκλυσης ή ενέσεις άμεσης έκλυσης. Η ομάδα των 28 γεννητόρων αποτελούνταν από ψάρια που αλι

Spermatogenesis enhancement in hatchery-produced greater amberjack (Seriola dumerili)

INTRODUCTION Greater amberjack Seriola dumerili (Risso, 1810) is a promising emerging aquaculture species thanks to its rapid growth and consumers’ appreciation. Wild-caught greater amberjack males reared in sea cages showed alteration of plasma sex steroid concentrations, high testicular apoptosis, reduced germ cell proliferation and low sperm quality. We report the effects of gonadotropin releasing hormone agonist (GnRHa) and human chorionic gonadotropin (hCG) administration on testis development and male germ cell proliferation in hatchery-produced greater amberjack. METHODS Four-year-old hatchery-produced greater amberjack males (F1 generation) reared in a sea cage in Salamina (Greece) were treated with GnRHa, either through EVAc implants (50 μg kg-1 body weight) or injections (20 μg kg-1 body weight), hCG (1000 IU kg-1 body weight) or were left untreated as controls. Two fish per group were treated in mid-May, when testes were in active spermatogenesis. Two weeks after treatments, fish were sacrificed and i) gonadosomatic index (GSI) was calculated as 100 × testis weight/body weight; ii) testis samples were fixed in Bouin’s solution and destined to histological analysis and to the immunodetection of the proliferating cell nuclear antigen (PCNA). The effects of the treatments on spermatogonial proliferation and germ cell progression towards meiosis was assessed through the count of the number of PCNA-positive single spermatogonia and the number of spermatocysts (PCNA-positive spermatogonial cysts + spermatocyte cysts). RESULTS & DISCUSSION The treatments resulted in an increase of GSI (untreated: 1.0 ± 0.2; GnRHa implant: 2.0 ± 1.4; GnRHa injection: 2.1 ± 0.8; hCG: 2.6 ± 0.7) and seminiferous tubule diameter (untreated: 142.5 ± 23.1 μm; GnRHa implant: 151.0 ± 14.7 μm; GnRHa injection: 196.1 ± 50.8 μm; hCG = 191.4 ± 8.3 μm). According to the subjective histological evaluation, testes of treated fish showed an increase of germinal epithelium height, larger lumina of seminiferous tubules and more abundant luminal spermatozoa compared with untreated controls. Fish treated with hCG showed the most dramatic changes, characterized by confluence of seminiferous tubules in large sperm masses in the internal testicular region. The hormone treatments resulted in both a decrease of proliferating single spermatogonia (untreated: 108.1 ± 1.3; GnRHa implant: 77.9 ± 47.4; GnRHa injection: 37.3 ± 9.2; hCG: 24.7 ± 21.3 cells/mm2) and an increase of PCNA-positive spermatocysts (untreated: 839.4 ± 12.8; GnRHa implant: 959.2 ± 30.8; GnRHa injection: 868.6 ± 379.5; hCG: 2074.0 ± 38.4 spermatocysts/mm2). In conclusion, all the tree treatments were effective in inducing testicular maturation through the stimulation of germ cell progression towards meiosis. Although the injection of hCG showed the most marked overall effects, GnRHa implantation was still able, after two weeks, to support both spermatogonial proliferation and progression towards meiosis, thus suggesting the capacity to sustain spermatogenesis over a longer period compared with the other two treatments. The project received funding from the ERA-NET Cofund BlueBio program (BESTBROOD project)