Influence of Docosahexaenoic and Eicosapentaenoic Acid Ratio and Temperature on the Growth Performance, Fatty Acid Profile, and Liver Morphology of Dusky Grouper (Epinephelus marginatus) (Teleostei: Serranidae) Juveniles.

An 8-week trial was performed to evaluate the influence of docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) ratios and temperature on the fatty acid (FA) metabolism and liver morphology of Epinephelus marginatus. A basal diet was manufactured, and DHA (D1:0.5%; D2:0.75%; D3:1%; and D4:1.5%) and EPA (D1:1.0%; D2:0.75%; D3:0.5%; and D4:0%) levels were added. E. marginatus were kept in twelve tanks with a lower temperature (LT, 23.17 ± 0.12 °C) and twelve with a higher temperature (HT, 28.63 ± 0.06 °C). The DHA/EPA ratio did not affect performance, regardless of the temperature, but the animals fed with the D4 diet showed better growth at HT. A higher lipid deposition and a large lipid vacuole area in the hepatocytes at HT were observed, regardless of the diet. Triacylglycerol (TG) in reflected the diet FA profile mainly in the muscle and liver, whereas the phospholipid (PL) was less influenced by the diet. The same DHA content in the TG fraction of muscle (D3 and D4) were observed at LT; however, only the DHA/EPA ratio of D4 could be differentially stored in the TG fraction of muscle (HT). Monounsaturated and polyunsaturated FA increased in the PL of the eyes at HT, whereas saturated FA was reduced in the TG and PL fractions at LT. These results evidence the importance of temperature and a balanced DHA/EPA ratio in the diet of marine fish.

Dynamics of ovarian maturation during the annual reproductive cycle of the endangered fish Steindachneridion parahybae (Siluriformes: Pimelodidae) in captivity.

To characterize the female reproductive biology of the endangered species Steindachneridion parahybae in captivity, the authors used the concentration of gonadal steroids and the oocyte development during the annual reproductive cycle (RC) and after artificial induced spawning (AIS) until 48 h. Three stages of gonadal maturation were identified, based on morphological and physiological features: early maturation or previtellogenic (PRV) oocyte, advanced maturation or vitellogenic (VTG) oocyte and regression (REG) or follicular atresia. They identified and characterized the following stages of germ cells: oogonia, perinucleolar and cortical alveoli, and VTG and atretic oocytes during RC. The oestradiol levels were higher in PRV than those in VTG and REG during the RC, whereas androgens showed higher levels of oestradiol in VTG than those in PRV and REG. The progestogen levels remained unchanged during the whole RC. The final oocyte maturation (FOM) was achieved after AIS and postovulatory follicles (POF) were identified. Plasma concentration of progestogens (17α,20ß-dihydroxy-pregnen-3-one and 17α-hydroxyprogesterone) increased considerably after AIS, remaining high up to 6 h after AIS, and progressively decreased over time after AIS. During RC, the lack of FOM and POFs reveals that captivity negatively impacts S. parahybae reproduction. Nonetheless, the VTG stage of oocytes, reached during RC, is suitable for ovulation induction with artificial hormone manipulation, enabling the reproduction of this species in captivity and being essential for the success of fish farming and/or fish conservation programmes (conservationist aquaculture).

Post-spawning feed deprivation effects on testicular and ovarian maturation in the neotropical cichlid fish Cichlasoma dimerus.

Many teleost fishes can withstand long feed deprivation periods, either due to an eventual lack of food or because of their behavior during reproduction and/or parental care. In this work, the effects of total food restriction on the oogenesis, spermatogenesis, and reproductive hormones of the neotropical cichlid fish Cichlasoma dimerus were studied. Specifically, different pairs were isolated after having a spawning event and were feed-deprived or daily fed for 3 weeks. After that period, gonadal histology, messenger levels of genes related to reproduction (gonadotropin-releasing hormone 1, gonadotropins, and insulin-like growth factor 1) and 11-ketotestosterone plasma levels were evaluated in both groups. Food restriction did not affect the reproductive axis in females since follicular maturation and gene expression showed no differences with respect to controls. However, in males, food restriction showed a stimulatory effect on the reproductive axis, reflected in a greater number of spermatozoa in their seminiferous lobes and spermatic ducts, and in an increase in follicle stimulating hormone messenger expression. Despite the negative effect reported for many fish species, C. dimerus seems to redirect their energy reserves towards gonadal development when faced with to a feed deprivation period.

Spawning induction and embryonic development of Salminus hilarii (Characiformes: Characidae).

The present study aimed to evaluate two different methods of artificial reproduction induction, and characterize the embryonic development of Salminus hilarii. Different than observed for other tropical fish species, artificial reproduction induction followed by hand-stripping of gametes was considered unfeasible for S. hilarii, as no gamete fertilization was observed. However, females that were induced and spawned naturally presented a fertilization rate of 65.64 ± 0.54%. With a mean temperature of 26.20 ± 0.90ºC it was possible to clearly distinguish a large perivitelline space at 14 min after fertilization (AF) and at 49 min AF more than 50% of the embryos presented two blastomeres, and these cleavages occurred until 1 h 54 min AF. The gastrula phase was characterized at 3 h 56 min AF, and blastopore closure was observed at 8 h 31 min AF. At 9 h 1 min, organogenesis started, with a clear distinction of the yolk mass, embryonic axis, cephalic and caudal regions; at 11 h 51 min AF the embryos already had advanced segmentation and a free tail. Total hatching occurred at 21 h 17 min and after opening the mouth, which occurred at 33 h 9 min, the larvae of S. hilarii presented a strong and characteristic cannibalism. This information can be considered fundamental to improving S. hilarii production in captivity and for collaboration with a conservation programme in the upper Tietê river basin.

Characterization of the gonadotropin-releasing hormone system in the Neotropical teleost, Steindachneridion parahybae during the annual reproductive cycle in captivity.

This study evaluated by immunohistochemical and Western blot methods, the distribution of two distinct gonadotropin-releasing hormones (GnRHs), corresponding to catfish GnRH (cfGnRH or GnRH1) and chicken-II GnRH (cGnRH-II or GnRH2), in Steindachneridion parahybae females in captivity, focusing these analyses on the reproductive cycle by semi-quantification of optical density (OD). Further, we found that the GnRH neuronal systems co-localized with their respective GnRH-associated peptides (GAPs). A group of neurons immunoreactive (ir) to GnRH1 were identified along the ventral region of the olfactory bulb (vOB) in the telencephalon (vTel) and in the main areas of the diencephalon (especially the medial basal hypothalamus, HBM), including fibers extending into the pituitary gland. In contrast, GnRH2 neurons were confined to the midbrain tegmentum, close to the ventricular surface, without projections to the pituitary gland. Moreover, a cfGAP (GnRH1)-specific band (9 kDa) was identified in the brain and pituitary gland, while a cGAP-II (GnRH2)-specific band (26 kDa) was observed only in the brain extract. During the reproductive cycle, GnRH1-ir presented greater OD values at the vitellogenic and regression stages than at the previtellogenic stage and after artificially induced to spawn. Larger GnRH2-ir neurons were observed during the reproductive cycle, but a higher OD was identified only in the regression stage compared with the other maturation stages. Finally, GnRH1 axons were found to be directed towards the pituitary, and this GnRH type, which is probably the hypophysiotropic form, can contribute to the reproductive dysfunction that occurs in S. parahybae females in captivity, whereas GnRH2 may act as a neuromodulator and/or neurotransmitter.

Characterization of gonadotropic cells during continuous and seasonal spermatogenesis of two freshwater fish species: a histochemical and immunohistochemical study.

This work describes gonadotropic (GtH) cells and their morphological and immunohistochemical changes during the spermatogenic cycle of Serrasalmus maculatus (continuous spermatogenesis) and Pimelodus maculatus (seasonal spermatogenesis). GtH cells, widely distributed in the proximal pars distalis of the adenohypophysis, were characterized as round-shaped cells with eccentric nucleus, and cytoplasm with basophilic secretory granules and a variable number of vacuoles for both species. Immunohistochemistry against ß-follicle-stimulating hormone (Fsh) and ß-luteinizing hormone (Lh) in adjacent sections showed two separated GtH-producing cell populations, and a third population where both GtHs are expressed in the same cell for both species. In the seasonal spermatogenesis of P. maculatus, GtH cells seemed to be more abundant during developing and spawning capable phases. In contrast, no cyclic changes were detected in the continuous spermatogenesis of S. maculatus, except for the strong immunoreaction for Fsh and Lh in males with intense spermiogenesis. We conclude that changes reported here might reflect the type of spermatogenic cycle (seasonal or continuous) which are under different regulatory mechanisms (environmental and internal cues) controlling the reproduction in these species.

The involvement of gonadotropins and gonadal steroids in the ovulatory dysfunction of the potamodromous Salminus hilarii (Teleostei: Characidae) in captivity.

Potamodromous teleosts that require migration to reproduce show dysfunctions that block ovulation and spawning while in captivity. To understand the physiological basis of these reproductive dysfunctions, follicle-stimulating hormone b subunit (fshb) and luteinizing hormone b subunit (lhb) gene expression analyses by real-time quantitative PCR, together with measurements of estradiol (E 2), 17α-hydroxyprogesterone (17α-OHP) and 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DHP) levels, were carried out throughout the reproductive cycle of the potamodromous Salminus hilarii. The following reproductive stages were evaluated in captive and wild females: previtellogenic (PV), advanced maturation/mature (AM) and regression/spent (REG/SPENT). In the wild females, fshb expression decreased from the PV to the AM stage, and the opposite pattern was detected for E 2, which increased from the PV to the AM stage. fshb was expressed at lower levels in captive than in wild females, and this difference did not change during the reproductive cycle. lhb expression also increased from the PV to the AM stage in both groups, but the wild females at the AM and REG/SPENT stages showed higher lhb expression levels than the captive females. The concentrations of 17α-OHP did not change during the reproductive cycle, and the levels were higher in the captive than in the wild females at all reproductive stages. 17α,20ß-DHP levels did not change between wild and captive females. However, in captive females, the transition from PV to AM stage was followed by an increase in 17α,20ß-DHP levels. These data indicate that dysfunctions in the gonadotropins and steroids synthesis pathways cause the ovulation failure in captive S. hilarii.

Gonadotropins and Growth Hormone Family Characterization in an Endangered Siluriform Species, Steindachneridion parahybae (Pimelodidae): Relationship With Annual Reproductive Cycle and Induced Spawning in Captivity.

The aim of this study was to identify and characterize pituitary cells of Steindachneridion parahybae females in captivity, highlighting the possible relationship with reproductive disorders at this level, since this species shows oocyte final maturation, ovulation and spawning dysfunction in captivity. The localization and distribution of growth hormone (GH), prolactin (PRL), somatolactin (SL), ß-luteinizing hormone (ß-LH), and ß-follicle stimulating hormone (ß-FSH) immunoreactive (-ir) cells in the adenohypophysis was studied by immunohistochemical and Western blot methods. In addition, cellular morphometric analyses and semi-quantification of ir-cells optical density (OD) during the annual reproductive cycle and after artificial induced spawning (AIS) were performed. Results showed that the distribution and general localization of pituitary cell types were similar to that of other teleost species. However, the morphometrical study of adenohypophysial cells showed differences along the reproductive cycle and following AIS. In general, females at the vitellogenic stage presented greater OD values for GH, PRL and SL than at other maturation stages (previtellogenic and regression stages), probably indicating an increased cellular activity during this stage. Conversely, ß-LH OD did not vary during the annual reproductive cycle. After AIS, ß-LH, SL and GH ir-cells showed an increase in OD values suggesting a possible involvement on oocyte final maturation, ovulation and spawning or a feedback control on the brain-pituitary-gonads axis. Reproductive dysfunction in S. parahybae females in captivity may be due to alteration of the synthesis pathways of ß-LH. In addition, GH family of hormones could modulate associated mechanisms that influence the reproductive status in this species.

The role of ovarian steroids in reproductive plasticity in Hoplias malabaricus (Teleostei: Characiformes: Erythrinidae) in tropical reservoirs with different degrees of pollution.

Chemicals in aquatic environments may lead to impairment of fish reproduction, but some species display plasticity levels to survive and reproduce in such localities. We investigated the reproductive cycle in a teleost species, Hoplias malabaricus, which inhabits a variety of environments with different degrees of pollution. Adult females were sampled at two locations in São Paulo State (Brazil), the reference Ponte Nova (PN) reservoir and the polluted Billings (BIL) reservoir. Metabolic and endocrine processes were analyzed throughout the annual cycle with special attention to ovarian histology, gonadosomatic index (GSI), fecundity, ovarian energetic substrates and plasma steroids analyses, in addition to physical and chemical analyses of the water. Pollutants were frequent in BIL but not in PN. The GSI, including the predominance of vitellogenic oocytes, was higher in spring and summer in both locations, but the oocyte recruitment dynamics was different. During winter, females from BIL presented vitellogenic oocytes and high levels of 11-ketotestosterone, which indicated precocity in the vitellogenic phase in relation to the females from PN. In animals from PN, high deposition of lipids occurred in the ovaries. However, plasma estradiol levels did not vary throughout the annual cycle. In animals from BIL, plasma estradiol levels peaked during the summer, but the ovarian lipid content remained unchanged throughout the year. The data suggest that the presence of pollutants cause various endocrine and metabolic responses and especially affects reproductive plasticity, which could explain why H. malabaricus survives under unfavorable environments.

Reproduction of the silver John dory Zenopsis conchifer (Actinopterygii: Zeiformes) based on virgin stock condition / Reprodução do peixe galo-de-profundidade Zenopsis conchifer (Actinopterygii, Zeiformes) baseada em um estoque virgem

This study describes the reproduction of the silver John dory, Zenopsis conchifer, based on data collected before its fishery exploitation in the southwestern Atlantic, Brazil, in the early 2000s. Biological material was collected through bottom-trawl surveys in 2001 and 2002 from depths of 100 to 600 m. A total of 1,056 individuals ranging from 57 to 504 mm Ls were analyzed. A sex ratio analysis showed that males were predominant among the shorter fishes (< 240 mm Ls) and females among the longer ones (> 320 mm Ls). Between 240 mm and 319 mm Ls sex-ratio was 1:1. The silver John dory is a multiple spawner. Spawning is protracted and more intensive during August and November, when both females and males in spawning capable actively spawning and in regression/regeneration stages were predominant. In this period average values of gonadosomatic index were also higher. For both sexes, the first maturation occurs at 220 mm Ls and at 240 mm Ls all fish can be considered adult. The recruitment apparently takes place from February to May. The length-weight relationships fitted to all specimens and for both sexes yieldedb coefficients of ∼2.7. The virgin stock ofZ. conchifer here characterized, may serve a basis for future studies on the effects of fisheries on the reproduction of the species.

O presente estudo descreve a reprodução do peixe galo-de-profundidade,Zenopsis conchifer, a partir de dados pretéritos a sua explotação pesqueira no Atlântico Sudoeste, Brasil, iniciada nos anos 2000. As coletas foram realizadas durante cruzeiros de prospecção pesqueira com arrasto-de-fundo em 2001 e 2002, em profundidades entre 100 e 600 m. Um total de 1056 exemplares (comprimento padrão entre 57 mm e 504 mm) foi examinado. A proporção sexual mostrou que machos predominam em comprimentos inferiores a 240 mm, que entre 240 mm e 319 mm a proporção é de 1:1 e que fêmeas predominam a partir de 320 mm. O peixe galo-de-profundidade apresenta desova múltipla, mais intensa entre agosto e novembro, quando predominam fêmeas e machos desovantes ativos e em regressão/regeneração. Neste período foram registrados os maiores valores do índice gonadossomático. Para ambos os sexos a primeira maturação se dá aos 220 mm de comprimento padrão e com 240 mm os indivíduos são considerados adultos. O recrutamento de jovens para o estoque adulto foi mais intenso entre fevereiro e maio. Os valores dos coeficientes b das relações comprimento peso ajustadas para todos os peixes e por sexo ficaram em torno de 2,7. As condições do estoque virgem de Z. conchiferaqui caracterizadas podem servir como um referencial para futuros estudos sobre os efeitos da pesca na reprodução da espécie.

The interrenal gland in males of the cichlid fish Cichlasoma dimerus: relationship with stress and the establishment of social hierarchies.

In teleosts, cortisol is the primary glucocorticoid secreted by the steroidogenic cells of the interrenal gland and an increase in its plasma concentration is a frequent indicator of stress. Cortisol has been postulated as an endogenous mediator involved in the regulation of reproduction and aggression related to social dynamics. The cichlid fish Cichlasoma dimerus, is a monogamous species that exhibits complex social hierarchies; males appear in one of two basic alternative phenotypes: non-territorial and territorial males. In this work, we postulated as a general hypothesis that the morphometry of the interrenal gland cells and the plasma levels of cortisol and 11-ketotestosterone (11-KT) are related to the social rank in adult males of C. dimerus. First, the location and distribution of the interrenal gland with respect to its context - the kidney - was studied. Plasma levels of cortisol and 11-KT in territorial and non-territorial males were established by ELISA. Finally, a morphometric analysis of steroidogenic and chromaffin cells of the interrenal gland was performed. Results showed that the interrenal gland was exclusively located in the posterior portion of the cephalic kidney. Non-territorial males presented a greater nuclear area of their steroidogenic cells. Additionally, plasma cortisol and 11-KT levels were lower and higher, respectively, in territorial males. Finally, plasma cortisol levels positively correlated with the nuclear area of interrenal steroidogenic cells. Thus, the interrenal gland, by means of one of its products, cortisol, may be fulfilling an important role in the establishment of social hierarchies and their stability.

Immunohistochemical study of pituitary cells in wild and captive Salminus hilarii (Characiformes: Characidae) females during the annual reproductive cycle.

Freshwater fish that live exclusively in rivers are at particular risk from fragmentation of the aquatic system, mainly the species that migrate upriver for reproduction. That is the case of Salminus hilarii, an important migratory species currently classified as "almost threatened" in the São Paulo State (Brazil), facing water pollution, dam construction, riparian habitat destruction and environmental changes that are even more serious in this State. Additionally, this species show ovulation dysfunction in captivity. Our studies focused on the identification and distribution of the pituitary cell types in the adenohypophysis of S. hilarii females, including a morphometric analysis that compares pituitary cells from wild and captive broodstocks during the reproductive annual cycle. The morphology of adenohypophysial cells showed differences following the reproductive cycle and the environment. In general, optical density suggested a higher cellular activity during the previtellogenic (growth hormone) and vitellogenic (somatolactin) stages in both environments. Additionally, the nucleus/cell ratio analysis suggested that growth hormone and somatolactin cells were larger in wild than in captive females in most reproductive stages of the annual cycle. In contrast, prolactin hormone showed no variation throughout the reproductive cycle (in both environments). Morphometrical analyses related to reproduction of S. hilarii in different environmental conditions, suggest that somatolactin and growth hormone play an important role in reproduction in teleost and can be responsible for the regulation of associated processes that indirectly affect reproductive status.

Dominance hierarchies and social status ascent opportunity: anticipatory behavioral and physiological adjustments in a Neotropical cichlid fish.

In this work we characterized the social hierarchy of non-reproductive individuals of Cichlasoma dimerus (Heckel, 1840), independently for both sexes, and its relationship to the opportunity for social status ascent. Female and male individuals who were located on the top rank of the social hierarchy, ascended in social status when the opportunity arose, therefore indicating that dominance is directly correlated with social ascent likelihood. Dominance was positively correlated with size in males but not in females, suggesting for the latter a relationship with intrinsic features such as aggressiveness or personality rather than to body and/or ovarian size. Physiological and morphometrical variables related to reproduction, stress and body color were measured in non-reproductive fish and correlated with dominance and social ascent likelihood. Dominance was negatively correlated with plasma cortisol levels for both sexes. No correlation with dominance was found for androgen plasma levels (testosterone and 11-ketotestosterone). No correlation was detected between dominance and the selected morphological and physiological variables measured in females, suggesting no reproductive inhibition in this sex at a physiological level and that all females seem to be ready for reproduction. In contrast, social hierarchy of non-reproductive males was found to be positively correlated with follicle stimulating hormone (FSH) pituitary content levels and gonadosomatic indexes. This suggests an adaptive mechanism of non reproductive males, adjusting their reproductive investment in relation to their likelihood for social status ascent, as perceived by their position in the social hierarchy. This likelihood is translated into a physiological signal through plasma cortisol levels that inhibit gonad investment through pituitary inhibition of FSH, representing an anticipatory response to the opportunity for social status ascent.