Rosa Zupa

Germ cell xenotransplantation is a promising tool for enhancing aquaculture production and supporting the conservation of declining fish species. A major limitation is the reliable identification of germ stem cells, which remains challenging due to the scarcity of validated molecular markers. This study assessed the suitability of different immunohistochemical approaches for identifying ovarian germ stem cells (OGSCs) in three Mediterranean species of commercial interest: European hake (Merluccius merluccius), meagre (Argyrosomus regius), and flathead grey mullet (Mugil cephalus). Among the antibodies tested (anti-OCT4, anti-VASA, anti-Sox2), only anti-VASA and anti-Sox2 labeled cells with morphological features consistent with OGSCs, although staining intensity, specificity, and cross-reactivity with early oocytes varied across species. In parallel, preliminary protocols for OGSC isolation and in vitro proliferation were developed for European hake and meagre. Enzymatic dissociation with collagenase, followed by Ficoll-Paque density separation and culture in Leibovitz’s L-15 medium supplemented with 2% fetal bovine serum at 25 °C, resulted in the highest OGSC recovery and detectable increases in proliferating cells. A sequential pre-plating step enhanced enrichment in meagre but caused substantial cell loss in hake. Overall, this study provides a comparative evaluation of female germline stem cell markers in these species, contributing to future surrogate reproduction and germplasm conservation strategies.

To overcome reproductive dysfunctions, recombinant gonadotropin hormones have been synthesised in different fish species. We were interested in gaining further insight into the effects of recombinant follicle-stimulating hormone (rFsh) in meagre Argyrosomus regius spermatogenesis and in improving the existing knowledge on the role of testicular apoptosis. Germ cell composition and testicular apoptosis were compared in pre-pubertal fish treated with rFsh (PreP-Fsh) and controls (PreP-C), and adult meagre with testes in the proliferative (Adult-Pro) and meiotic (Adult-Meio) spermatogenesis phases. A lower density of committed spermatogonia was observed in PreP-Fsh compared with PreP-C and in Adult-Meio compared with Adult-Pro. In pre-pubertal fish and in Adult-Pro, apoptosis affected mainly individual germ cells and Sertoli cells. In Adult-Meio, a high number of cysts containing apoptotic germ cells was observed, and apoptosis often involved entire clones of germ cells. rFsh reduced testicular apoptosis in pre-pubertal fish, whereas the advancement of spermatogenesis in adults was associated with an increased density of apoptotic cells. The present study corroborated previous observations on the effects of rFsh on spermatogenesis in pre-pubertal meagre and provided further insights on the changing role of apoptosis in the pre-pubertal phase versus different phases of spermatogenesis.

Cyprinids include some of the most widely farmed freshwater species. The aim of this study was to assess the incidence of skeletal malformations in carp species reared in semi-intensive systems in Albania: common carp (Cyprinus carpio), silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idella) and bighead carp (Hypophthalmichthys nobilis). The common carp fingerlings had a mean total length of 28.9 ± 5.0 mm; the frequencies of individuals with at least one anomaly and at least one severe anomaly were 79.2% and 43.4%, respectively. The silver carp juveniles had a mean total length of 21.6 ± 2.1 mm; the frequencies of individuals with at least one anomaly and one severe anomaly were 93.1% and 57.5%, respectively. The grass carp fry had a mean total length of 33.5 ± 2.6 mm; all the analyzed specimens showed almost one anomaly and 86.4% showed at least one severe anomaly. The bighead carp juveniles had a mean total length of 34.4 ± 5.7 mm; the frequencies of individuals with at least one anomaly and at least one severe anomaly were 95.0% and 62.5%, respectively. The development of a more suitable feeding protocol for herbivorous species and the setting up of more efficient broodstock management protocols are suggested to reduce the high incidence of skeletal malformations.

The meagre Argyrosomus regius (Asso, 1801) is a promising aquaculture species that shows reproductive dysfunctions when reared in tanks. The aim of this study was to assess the capacity of meagre, reared in cages under routine farming conditions, to mature gonads and reproduce spontaneously. Meagre adults, reared in a fish farm located in the Gulf of Taranto (Italy), were sampled from March to July 2021. The gonadosomatic index and sex steroid plasma concentrations increased from March–April to June, and then decreased in July. In March–April, most of the females showed perinucleolar or cortical alveoli oocytes as the most advanced stages in the ovaries, and most of the males had testes at early spermatogenesis stage. In June, most of the sampled females had oocytes at late vitellogenesis or early post-vitellogenesis stages, and males had seminiferous tubules filled with spermatozoa. In July, most of the females had signs of previous spawning, and males showed scarce amounts of luminal spermatozoa. The present study demonstrated the capacity of meagre, reared in sea cages under commercial conditions, to carry out gametogenesis and spontaneously release gametes. Meagre reproduction, under routine farming conditions, may represent an opportunity for the expansion of meagre aquaculture production.

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.

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.

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.

The greater amberjack, Seriola dumerili (Risso, 1810), is a promising candidate for the diversification of European aquaculture production, but inconsistent reproduction in captivity prevents commercial production. Recent studies showed that greater amberjack confined in sea cages exhibited scarce gonad development and early interruption of gametogenic activity during the reproductive season. The aim of the present study was to improve our understanding of the observed impairment of spermatogenesis. Adult wild and captive-reared males were sampled during 3 different phases of the reproductive cycle: early gametogenesis (EARLY; late April to early May), advanced gametogenesis (ADVANCED; late May to early June), and spawning (SPAWNING; late June to July). Spermatogonial stem cells and proliferating germ cells were identified through the immunohistochemical localization of Pou5f1 and proliferating cell nuclear antigen, respectively. Apoptotic germ cells were identified throughout the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling method. Sperm quality of captive-reared fish was evaluated using computer-assisted sperm analysis. Captive-reared males exhibited seminiferous lobules of a smaller diameter, a precocious and progressive decrease of spermatogonial mitosis, and a high level of apoptosis at the beginning of the reproductive season, concomitant with a many-fold higher 17β-estradiol plasma concentration. The motile spermatozoa percentage of captive greater amberjack was lower than in other teleosts, and a drastic decrease of spermatozoa motility duration, velocity, and ATP content occurred along the reproductive season. An abnormal increase of sperm concentration as well as an increase of dead spermatozoa occurred during the SPAWNING phase, probably because of lack of sperm hydration and ejaculation and consequent sperm ageing. The present study demonstrates the extreme susceptibility of greater amberjack to rearing stress and underscores the need for improvement of the rearing and handling procedures to ameliorate gametogenesis dysfunctions in commercial aquaculture production.

The greater amberjack Seriola dumerili is a large teleost fish with rapid growth and excellent flesh quality, whose domestication represents an ambitious challenge for aquaculture. The occurrence of reproductive dysfunctions in greater amberjack reared in captivity was investigated by comparing reproductive development of wild and captive-reared individuals. Wild and captive-reared breeders were sampled in the Mediterranean Sea during 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). Fish reproductive state was evaluated using the gonado-somatic index (GSI), histological analysis of the gonads and determination of sex steroid levels in the plasma, and correlated with leptin expression in the liver and gonad biochemical composition. The GSI and sex steroid levels were lower in captive-reared than in wild fish. During the ADVANCED period, when the wild greater amberjack breeders were already in spawning condition, ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes and spermatogenic activity ceased in the testes of half of the examined males. During the SPAWNING period, all captive-reared fish had regressed gonads, while wild breeders still displayed reproductive activity. Liver leptin expression and gonad proximate composition of wild and captive greater amberjack were similar. However, the gonads of captive-reared fish showed different total polar lipid contents, as well as specific lipid classes and fatty acid profiles with respect to wild individuals. This study underlines the need for an improvement in rearing technology for this species, which should include minimum handling during the reproductive season and the formulation of a specific diet to overcome the observed gonadal decrements of phospholipids, DHA (22:6n-3) and ARA (20:4n-6), compared to wild breeders.