Ontogenetic change in the body length–mass relationship concomitant to the onset of vitellogenesis in female blackmouth catshark Galeus melastomus (Chondrichthyes: Scyliorhinidae)

The examination of the total length (L) vs. body mass (W) relationship in the blackmouth catshark from the southern Adriatic Sea showed the occurrence of two development phases or growth stanzas in females. The passage from the first stanza (L range: 22.8-37.4cm) to the following one (L range: 39.7-51.4cm) was marked by an inflection in the power equation correlating body mass to total length, occurring at about 38.6cm of length (change point). After the change point, growth became positively allometric. This catshark is a lecitotrophic oviparous continuous spawner, and follicles in different stages of development are simultaneously present in the ovary. The histological analysis of the ovary showed that the smallest vitellogenic follicles were about 2mm in diameter; the diameter of the largest observed oocyte was 18mm. The change point occurred slightly before the onset of vitellogenesis (smallest vitellogenic female L=41.0cm) and appeared to be related to the activation of the reproductive axis

Atresia of ovarian follicles in fishes, and implications and uses in aquaculture and fisheries

Atresia of ovarian follicles, that is the degenerative process of germ cells and their associated somatic cells, is a complex process involving apoptosis, autophagy and heterophagy. Follicular atresia is a normal component of fish oogenesis and it is observed throughout the ovarian cycle, although it is more frequent in regressing ovaries during the postspawning period. An increased occurrence of follicular atresia above physiological rates reduces fish fecundity and even causes reproductive failure in both wild and captive-reared fish stocks, and hence, this phenomenon has a wide range of implications in applied sciences such as fisheries and aquaculture. The present article reviews the available literature on both basic and applied traits of oocyte loss by atresia, including its morpho-physiological aspects and factors that cause a supraphysiological increase of follicular atresia. Finally, the review presents the use of early follicular atresia identification in the selection process of induced spawning in aquaculture and the implications of follicular atresia in fisheries management

Male germ cell proliferation and apoptosis in sexually immature meagre Argyrosomus regius (Asso, 1801) treated with recombinant follicle stimulating hormone

The meagre Argyrosomus regius (Asso, 1801) is a marine fish species that has an increasing aquaculture production in Europe. Lowering the age at maturity of hatchery-produced juveniles would support meagre aquaculture by reducing time between generations in selective breeding programs and reducing industrial costs for broodstock maintenance. The aim of this work was to assess the effects of a treatment with recombinant follicle stimulating hormone (rFsh), produced in ovarian cells of Chinese hamsters, on male germ cell proliferation and apoptosis in sexually immature meagre. The rFsh-treated fish had higher gonadosomatic index, larger seminiferous tubules, more abundant luminal spermatozoa, a lower density of anti-PCNA positive single A spermatogonia, a higher density of anti-PCNA positive spermatocysts and a lower incidence of germ cell apoptosis than control groups. The present study demonstrated the effectiveness of the produced rFsh in stimulating testis development and spermatogenesis in pre-pubertal meagre. Moreover, the rFsh treatment proved to be highly efficient in removing the apoptotic block of spermatogenesis observed in juvenile meagre, allowing spermatogonial survival and progress towards meiosis. The administration of rFsh did not stimulate spermatogonial self-renewal, a process whose control still needs to be elucidated.info:eu-repo/semantics/publishedVersio

Differences in macroelements, trace elements and toxic metals between wild and captive-reared greater amberjack (Seriola dumerili) from the Mediterranean Sea

Despite its legislative regulation and control, the quality and safety of aquatic products is somewhat questioned due to the potential bioaccumulation of pollutants. The elements (Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Sr, V and Zn) were determined in the liver and muscle of wild and captive-reared Seriola dumerili with the aim of studying possible differences between origins, and sex-related variations. Additionally, the dietary intake of these elements derived from its consumption was also evaluated. Most of the elements and metals analyzed were accumulated to a higher extent in the liver of wild specimens whereas lower differences were observed in the muscle. Overall, the elements and metal composition of wild females strongly differed from that of captive-reared specimens probably related to the mobilization of nutrients for the spawning season in wild mature females, which were greater than their captive-reared counterparts

Rearing in captivity affects fish spermatogenesis through changes in germ cell proliferation and apoptosis

Spermatogonial proliferation and loss of germ cells via apoptosis are critical processes during fish spermatogenesis. The correct balance between spermatogonial self-renewal and differentiation towards meiosis determines the eventual sperm output. Moreover, apoptosis, a form of programmed cell death, plays a role in maintaining a correct Sertoli/germ cells ratio, limiting germ cell population and preventing maturation of aberrant germ cells. These processes are controlled by pituitary gonadotrophins- follicle-stimulating hormone (FSH) and luteinizing hormone (LH)- whose release is stimulated by gonadotrophin-releasing hormone (GnRH). Most male fish reared in captivity display reproductive dysfunctions which are supposed to be caused by an insufficient pituitary stimulation by GnRH, and the consequent inadequate gonadotrophin release, as a result of the combination of captivity-induced stress and lack of adequate natural spawning conditions. The effects of rearing in captivity on large pelagic fish (Atlantic bluefin tuna Thynnus thynnus; greater amberjack Seriola dumerili) germ cell proliferation and apoptosis are herein argued. In captive-reared Atlantic bluefin tuna, a delay of spermatogonial proliferation and an increase of germ cells apoptosis (Fig. 1) were associated to low 11-Ketotestorone (11-KT) levels. In captive-reared greater amberjack, a high level of germ cell apoptosis at the beginning of the breeding phase was observed, along with a progressive decrease of germ cell proliferation during the reproductive season. Both in Atlantic bluefin tuna and greater amberjack, the observed gametogenesis dysfunctions eventually resulted in low quality sperm production and, in Atlantic bluefin tuna, they were alleviated by the administration of a GnRH agonist (GnRHa). Proper rearing practises, including handling procedures that minimize stress, along with hormonal therapies, are recommended to ameliorate spermatogenesis dysfunctions

Meagre Argyrosomus regius (Asso, 1801) Stem Spermatogonia: Histological Characterization, Immunostaining, In Vitro Proliferation, and Cryopreservation

The meagre, Argyrosomus regius, is a valued fish species of which aquaculture production might be supported by the development of a stem germ cell xenotransplantation technology. Meagre males were sampled at a fish farm in the Ionian Sea (Italy) at the beginning and end of the reproductive season. Small and large Type A undifferentiated spermatogonia were histologically identified in the germinal epithelium. Among the tested stemness markers, anti-oct4 and anti-vasa antibodies labeled cells likely corresponding to the small single Type A spermatogonia; no labeling was obtained with anti-GFRA1 and anti-Nanos2 antibodies. Two types of single A spermatogonia were purified via density gradient centrifugation of enzymatically digested testes. Testes from fish in active spermatogenesis resulted in a more efficient spermatogonial stem cell (SSC) yield. After cell seeding, meagre SSCs showed active proliferation from Day 7 to Day 21 and were cultured up to Day 41. After cryopreservation in dimethyl-sulfoxide-based medium, cell viability was 28.5%. In conclusion, these results indicated that meagre SSCs could be isolated, characterized, cultured in vitro, successfully cryopreserved, and used after thawing. This is a first step towards the development of a xenotransplantation technology that might facilitate the reproduction of this valuable species in captivity

Reliability of melanomacrophage centres as indicators of stress response in teleost fishes

Melanomacrophage centres (MMCs) are aggregates of pigmented phagocytes, characterized by heterogeneous inclusions and located in hemolymphopoietic organs of various non-mammalian vertebrates [1]. In the present work, data from published and ongoing studies on MMCs were analysed in order to get insights on MMC reliability as response biomarkers to stress and environmental pollution. Liver samples from 31 wild and captive reared Atlantic bluefin tuna (Thunnus thynnus) [2, 3] and from 90 wild European anchovies (Engraulis encrasicolus) caught in areas differently exposed to industrial and agricultural pollutants [4], and liver and spleen samples from 47 wild and captive reared greater amberjack (Seriola dumerili) [5, unpublished data] were fixed in 10% buffered formalin and embedded in paraffin wax. Deparaffinized sections were stained with haematoxylin-eosin; Mallory’s basic fucshin (Merck) and Perls VanGieson (Bio-Optica) stainings were used to identify lipofuscin–ceroids and ferric iron respectively; peroxidase detection was performed by a Leukocyte Peroxidase kit (Sigma). The terminal deoxynucleotidyl transferase-mediated d’UTP nick-end labelling (TUNEL) method was used to identify apoptotic cells and the immunohistochemical detection of cytochrome P450 monooxygenase 1A (CYP1A) was performed by means of polyclonal antibodies anti-fish CYP1A (Biosense Laboratories). Lipofuscin–ceroids and ferric iron were detected in MMCs of all the three examined species, whereas peroxidase was mainly detected in free macrophages. In Atlantic bluefin tuna, a high density of MMCs and liver apoptotic cells, and a strong CYP1A immunostaining were observed in young individuals reared in the central Adriatic Sea compared with adults caught from the wild or reared in the western Mediterranean. In European anchovy, a high density of MMCs and a strong CYP1A immunostaining were observed in fish sampled in the Gulf of Gela, a marine area dramatically affected by environmental pollution. In greater amberjack, MMC density was higher in spleen than in liver sections. Confinement in captivity did not affect MMC density, whereas differences were found between males and females and among fish in different reproductive conditions. The present study confirms that MMCs represent a useful biomarker of fish exposure to environmental pollution; however, sex and reproductive state may affect MMC density, possibly leading to data misinterpretations

Proliferation and Apoptosis of Cat (Felis catus) Male Germ Cells during Breeding and Non-Breeding Seasons

The domestic cat (Felis catus) is a seasonal-breeding species whose reproductive period starts when the day length increases. Since the existing information on cat spermatogenesis is limited and somewhat contradictory, in the present study, germ cell proliferation and apoptosis in feral adult tomcats orchiectomized during reproductive (reproductive group, RG; February–July) and non-reproductive (non-reproductive group, NRG; November and December) seasons were compared. Cross-sections taken from the middle third of the left testis were chemically fixed and embedded in paraffin wax. Histological sections were processed for the immunohistochemical detection of proliferating germ cells (PCNA) and for the identification of apoptotic cells (TUNEL method). The percentage of PCNA-positive spermatogonia was higher in the RG than in the NRG. On the contrary, germ cell apoptosis was higher in the NRG than in the RG. Our results confirm that cat spermatogenesis is modulated on a seasonal basis and suggests that spermatogenesis control involves changes in germ cell proliferation and apoptosis according to a common paradigm of seasonally breeding species

OOGENESIS IN WILD AND REARED GREATER AMBERJACK SERIOLA DUMERILI (RISSO, 1810)

Introduction The incorporation of new species in the aquaculture industry necessitates to control the reproductive function in captivity and to produce high numbers of high-quality eggs. Greater amberjack Seriola dumerili (Risso, 1810) caught from the wild and reared in captivity have been shown not to develop further than early vitellogenesis or if they did complete vitellogenesis, they failed to undergo oocyte maturation and required exogenous hormonal therapies to induce ovulation and spawning (Mylonas et al., 2004).The present work represents an overview of the results obtained in a study on the oogenesis of wild and captive-reared greater amberjack carried out within the EU FP7 project Diversify (Zupa et al, 2017; Pousis et al., 2018, 2019). Material and Methods Twenty-one wild and twelve captive-reared greater amberjack females were sampled during 2014, 2015 and 2016 at three different phases of the reproductive cycle: early gametogenesis (EARLY), late April-early May (wild fish = 5; captive-reared fish = 4); advanced gametogenesis (ADVANCED), late May-early June (wild fish = 4; captive-reared fish = 4); spawning (SPAWNING), late June-early July (wild fish = 12; captive-reared fish = 4). Wild fish were sampled on board a professional purse-seine fishing vessel operating around the Pelagie Islands (Sicily, Italy); captive-reared individuals belonged to a broodstock captured as juveniles and moved to a sea cage of Argosaronikos Fishfarming S.A. (Salamina Island, Greece). For each fish, biometric data (fork length, FL, in cm; body mass, BM, in kg; testis mass, TM, in g) were registered and gonadosomatic index (GSI = 100 × TM/BM) was calculated. Liver samples were store at -80°C and subsequently used for the analysis of vitellogenin (vtga, vtgb and vtgc) expression through RT-PCR. Ovary samples were used for histological analysis and for vitellogenin receptor (vtgr and lrp13) expression analysis through RT-PCR. Blood samples were centrifuged and plasma was stored at -20°C for the analysis of testosterone, 17β-estradiol and 17,20β-dihydroxypren-4-en-3-one by ELISA assays. Results and Discussion The GSI and all the sex steroid plasma levels were lower in captive-reared fish. During the EARLY phase, wild and captive-reared fish displayed perinucleolar or early vitellogenesis as the most advanced oocyte stage. During the ADVANCED phase, when the wild greater amberjack breeders were already in spawning condition (Fig. 1a), ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes (Fig. 1b). During the SPAWNING period, all captive-reared fish had regressed ovaries, while wild breeders still displayed oocytes at late vitellogenesis and maturation stages as well as postovulatory follicles. The expression levels of vtga, vtgb and vtgc did not differ significantly between captive-reared and wild females. Ovarian vtgr and lrp13 transcription was more active during early gametogenesis, suggesting that vitellogenin receptor transcripts were synthesized by previtellogenic oocytes and remained in the cellular mRNA pool until oocytes resumed meiosis and entered vitellogenesis. A reduced vtgr and lrp13 transcription was observed in captive-reared compared wild greater amberjack during the EARLY phase. The observed reproductive dysfunction, leading to oocyte atresia and reduced gonadosomatic index, arose during the early phase of oogenesis, when transcription of vitellogenin receptor genes appeared to be reduced, and did not appear to be associated to a lower liver capacity to synthesize the egg yolk precursors. Severe reproductive dysfunctions were observed also in males of the same broodstock and involved low sex steroid plasma concentrations and precocious cessation of spermatogenesis (Zupa et al., 2017). Preliminary data obtained within the H2020 project NewTechAqua indicate that hatchery-produced greater amberjack reared in sea cages in Salamina (Greece) have similar GSI compared with wild fish sampled in the same period of the reproductive cycle (early June 2021). Although further analyses are required, the available data indicate that hatchery-produced individuals might be less affected by captivity-induced stress than wild-caught breeders. Financial grant provided by the European Union ́s Programmes FP7 (GA 603121, DIVERSIFY) and H2020 (GA 862658, NewTechAqua). References Mylonas, C.C., Papandroulakis, N., Smboukis, A., Papadaki, M., Divanach, P. 2004. Induction of spawning of cultured greater amberjack (Seriola dumerili) using GnRHa implants. Aquaculture, 237: 141-154. Pousis, C., Mylonas, C. C., De Virgilio, C., Gadaleta, G., Santamaria, N., Passantino, L., Zupa, R., Papadaki, M., Fakriadis, I., Ferreri, R., Corriero A. 2018. 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. Aquaculture Research, 49: 243-252. Pousis, C., Rodríguez, C., De Ruvo, P., De Virgilio, C., Pérez, J.A., Mylonas, C. C., Zupa, R., Passantino, L., Santamaria, N., Valentini, L., Corriero A. 2019. Vitellogenin receptor and fatty acid profiles of individual lipid 1 classes of oocytes from wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle. Theriogenology, 140: 73-83. Zupa, R., Rodríguez, C., Mylonas, C. C., Rosenfeld, H., Fakriadis, I., Papadaki, M., Pérez, J. A., Pousis, C., Basilone, G., Corriero, A. 2017a. Comparative study of reproductive development in wild and captive-reared greater amberjack Seriola dumerili (Risso,1810). PLoS ONE 12(1): e0169645

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)