Increase in milt production by hormonal treatment in the pejerrey fish Odontesthes bonariensis (Valenciennes 1835)

In spite of interest in the cultivation of the pejerrey fish Odontesthes bonariensis (Cuvier & Valenciennes 1835), there are few studies on subjects required to advance this activity. One of the problems is the synchronization of female and male maturation to provide eggs and sperm for larval production. The low volume of expressible milt, either in wild or culture fish, is a major problem. The aim of this work was to study the effectiveness of the administration of different hormones on sperm production in pejerrey. Milt production was enhanced by the injection of human chorionic gonadotropin (hCG) (16.7‐fold increase, 625 IU kg^?1), carp pituitary extracts (13.5‐fold increase, 30 mg kg^?1), salmon pituitary extracts (12.8‐fold increase, 30 mg kg^?1), salmon‐type gonadotropin‐releasing hormone analogue (GnRH) (16.7‐fold increase, 10 μg kg^?1) and mammalian‐type GnRH analogue (10.8‐fold increase, 20 μg kg^?1). Sperm concentration, motility and the fertilization rate were not statistically different compared with control groups. It was also demonstrated that sperm could be obtained off‐season. Taken together, hCG is recommended to stimulate pejerrey spermiation because it is effective in low doses is inexpensive and is widely available.     

Spawning induction of pejerrey Odontesthes bonariensis in captivity using sustained‐release gonadotropin releasing hormone agonist implants

The aim of this study was to induce and synchronize spawning of pejerrey Odontesthes bonariensis (Valenciennes, 1835), using gonadotropin releasing hormone agonist (GnRHa) implants. In the first experiment, the ovarian condition was assessed by ovarian biopsies and the measurement of the genital pore width (GPW). Females having the leading clutch of oocytes with a diameter of around 800–900 μm and a GPW between 4.5 and 5.5 mm were treated with GnRHa implants. Eighty per cent of females spawned between 2 and 9 days after treatment, 12 days earlier than 20% of the fish in the control group that presented signs of spawning activity. In order to avoid any possible ovarian injury and/or stress by the catheterization procedure, in a second experiment, females were selected only by visual inspection of the abdomen and GPW measurement. As in experiment 1, 80% of females spawned between 2 and 8 days after treatment, 8 days earlier than 30% of the fish that spawned in the control group. In both experiments, fertilization and hatching success were similar between control and GnRHa‐treated groups. These results clearly demonstrated that GnRHa implantation can advance and synchronize ovulation and spawning in pejerrey without affecting egg quality.     

Characterization of three GnRH cDNA sequences in the pejerrey fish Odontesthes bonariensis

In this work we have characterized cDNAs fragments of GnRH preprohormones by 3′RACE. This characterization confirms the presence and expression of three GnRH forms in brain tissue and constitutes an initial step in the study of the physiological role of GnRHs during the sexual differentiation, maturation and reproduction of pejerrey.     

Molecular cloning of SOX9, DMRT1 and SF1 cDNA partial sequences in the pejerrey fish Odontesthes bonariensis (Atheriniformes)

Three different genes known to be involved in the mammalian sex-determining pathway, SOX9, DMRT1 and SF1, were characterized in a remarkable temperature sex determining species, the pejerrey Odontesthes bonariensis.     

Distinct expression of GnRH genes in the red seabream brain

This paper reports the molecular cloning of a cDNA encoding the precursor of seabream gonadotropin-releasing hormone (prepro-sbGnRH) and the localization of salmon GnRH (sGnRH) and seabream GnRH (sbGnRH) expressing neurons in the brain of the red seabream (Pagrus major). The cloned prepro-sbGnRH cDNA has a 285 bps open reading frame encoding a 23 amino acid signal peptide, a 10 amino acid sbGnRH, the cleavage site (Gly-Lys-Arg), and a 59 amino acid GnRH-associated peptide. The expression of sGnRH and sbGnRH peptides, and prepro-sGnRH and prepro-sbGnRH mRNA were studied using immunocytochemistry and non-radioactive in situ hybridization, respectively. We found cell bodies that reacted positively with both the sGnRH cRNA probe and anti-sGnRH serum, but not with the sbGnRH cRNA probe or anti-sbGnRH serum in the ganglion of the terminal nerve. Cell bodies that reacted positively with the sbGnRH cRNA probe, anti-sbGnRH serum, and anti-sGnRH serum, but negatively with the sGnRH cRNA probe were found in the preoptic area (POA). Immunocytochemistry showed that a distinct bundle of axons arises in the POA which projected to the pituitary gland. These results suggest that sbGnRH is the most relevant hypophysiotropic form of GnRH.     

Muscular hypertrophy and growth‐promoting effects in juvenile pejerrey (Odontesthes bonariensis) after oral administration of recombinant homologous growth hormone obtained by a highly efficient refolding process

Growth hormone (GH) can be orally administrated to fish in order to increase growth rates. Fish growth is characterized by the hyperplasia and hypertrophy of muscle fibre throughout adult life. In this respect, GH could affect directly and indirectly (by growth and metabolic factors) the development and growth of muscle fibres. Recombinant pejerrey GH (r‐pjGH) was expressed in Escherichia coli and refolded in a highly efficient batch dilution system, obtaining 0.1 g L^?1 of hormone without protein precipitation during the refolding procedure. Orally administered hormone to pejerrey produced a 30% increase in mean weight and stimulated liver insulin‐like growth factor type I (IGF‐I) mRNA expression after 1 month of treatment. Histological analyses showed that muscle growth was generated mainly by hypertrophy of the fibres. A higher r‐pjGH dose increased muscle fibre hypertrophy but somatic growth was negatively affected probably due to a reduced capacity of generating new fibres.     

Effects of stocking density and natural food availability on the extensive cage culture of pejerrey (Odontesthes bonariensis) in a shallow Pampean lake in Argentina

An experiment was conducted for 80 days at La Salada de Monasterio Lake (Buenos Aires, Argentina) to assess the effect of stocking density and natural food availability on the growth and production of zooplanktivorous juveniles of pejerrey (Odontesthes bonariensis) in extensive cage culture. Ten cages were installed and stocked with 33‐day‐old fish, at three density treatments: 25, 50 and 75 ind. m^?3. Zooplankton was analysed in terms of abundance, biomass and diversity considering three size classes. Caged pejerrey diet was assessed regularly. The pejerrey gut contents composition was clearly different from cage zooplankton, showing a trend to contain bigger components. Reared fish exhibited a tendency to diversification of the diet and to change the mean prey size depending on fish length and stocking density. Regression models showed a positive and direct effect of the bigger zooplankton biomass on fish growth rates, which were also inversely affected by the availability of smaller zooplankton. The results demonstrate that stocking density and available zooplankton, in both quantity and quality, are key factors in regulating extensive cage culture of pejerrey. Handling stocking densities in a dynamic way it is possible to maximize fish growth, biomass harvest or fish number according to the goals of production.     

Extensive cage culture of pejerrey (Odontesthes bonariensis) in a shallow pampean lake in Argentina

Pejerrey is an important zooplanktivorous native fish of the Argentinean inland waters. It has been traditionally propagated for stocking purposes by relatively costly semi‐intensive and intensive methods. In this study, we evaluated the implementation of an extensive culture method by using floating cages in a shallow pampean lake. Four cages were installed in the Lacombe Lake and stocked with juveniles (16.24 ± 1.69 mm length) at 50 fish m^?3 density for growing until the size of 150 mm, which is considered as a suitable size for stocking. Throughout the experiment, the temperature ranged between 10 and 26 °C and the zooplankton biomass ranged between 12 and 3269 μg dw L^?1. The growth patterns in the length were similar in the four cages and directly related to the lake thermal conditions and zooplankton availability. The average final length after 315 days was 154.4 ± 8.8 mm. The survival rates ranged between 53.5% and 64.7% during the first 110 days and 11.1–25.7% at termination. Growth rate for the first 2 months was the highest documented for pejerrey culture. This simple technique offers the possibility to produce juvenile pejerrey at a low cost and provides the alternative of reinforcing the natural populations with fish already adapted to the natural environmental conditions.     

Close association of gonadotropin-releasing hormone fibers and gonadotropin, growth hormone, somatolactin and prolactin expressing cells in pejerrey, Odontesthes bonariensis

The purpose of this study was to determine if there is any association between immunoreactive (ir) gonadotropin-releasing hormone (GnRH) fibers with different pituitary endocrine cell types in the pejerrey, Odontesthes bonariensis. Using a monoclonal antibody raised against mammalian GnRH (mGnRH) (LRH13), ir-GnRH fibers were observed passing through the pituitary stalk and reaching the three areas of the pituitary gland: rostral (RPD) and proximal pars distalis (PPD) and pars intermedia (PI). Double labeled immunocytochemistry showed ir-GnRH fibers in close association with prolactin (PRL)-producing cells in the RPD, growth hormone (GH)-producing cells in the PPD, gonadotropin (GtH)-producing cells in the PPD and the external border of the PI, and with somatolactin (SL)-producing cells in the PI. Our results show, direct morphological evidences of a close association of GnRH fibers with GH, PRL, GtH and SL-expressing cells. These results would suggest that GnRH has a broad role in the regulation of the secretion of different pituitary hormones.     

mRNA expression of GnRH variants and receptors in the brain,pituitary and ovaries of pejerrey (<Emphasis Type="Italic">Odontesthes bonariensis</Emphasis>) in relation to the reproductive status

The present study examined the differential mRNA expression levels of three forms of GnRH (sGnRH, pjGnRH and cGnRH-II) and two forms of GnRH receptor (pjGnRH-R I and pjGnRH-R II) in the brain, pituitary, and ovaries of pejerrey in relation to the reproductive status. The analysis revealed the presence of significant amounts of mRNA of the three GnRH forms while the ovaries showed only two (sGnRH and pjGnRH). The GnRH receptor II was found ubiquitously in the brain, pituitary, and ovaries while the form I was detected only in the brain. The levels of pjGnRH mRNA in the brain and pjGnRH-R II in the pituitary gland varied in correlation with the ovarian condition. However, brain sGnRH and pjGnRH-R I mRNA levels reached a maximum during early stages of ovarian development. In contrast, the brain levels of cGnRH-II mRNA showed no variation. The present study also shows a good correlation of ovarian sGnRH and pjGnRH-R II mRNA levels with the reproductive condition, suggesting that these molecules are may be involved in the regulation of pejerrey ovarian function.     

Leucocyte profile and growth rates as indicators of crowding stress in pejerrey fingerlings (Odontesthes bonariensis)

Crowding is one of the most common stressors found in intensive aquaculture, compromising growth rates and immune function. Plasmatic cortisol is a classic stress biomarker for fish, but its quantification is expensive and demands blood volumes that small individuals do not provide, constraining the usage of this technique to assess stress in fingerlings. The leucocyte profile is an alternative methodology to quantify stress with reduced costs and volumes of blood. Stress conditions promote neutrophilia and lymphocytopenia as response to elevated glucocorticoids levels. Considering the difficulties to assess stress imposed by intensive fish farming using measurement of glucocorticoid hormones, this study aimed to evaluate the stress‐induced changes in leucocyte profiles and growth rates imposed by crowding in fingerlings of Odontesthes bonariensis, a promising South‐American candidate for freshwater aquaculture. To meet these objectives, fingerlings (initial weight 0.05 ± 0.06 g and length 1.68 ± 0.13 cm) were reared for 45 days under three rearing densities (1, 5 and 10 fingerlings L^?1). At the end of this period, fish were anaesthetized and euthanized to obtain the leucocyte profile, neutrophil:lymphocyte ratio (N:L) and growth rate. Increasing density promoted: significant reduction in growth (final length, weight and specific growth rate); neutrophilia and lymphocytopenia; and increased N:L ratio. Concluding, the tested rearing densities imposed distinct levels of stress characterized by different N:L ratio, demonstrating that the leucocyte profile is a reliable alternative to measure stress levels in O. bonariensis fingerlings and probably in other fish species.     

Molecular cloning of cDNAs encoding FSH-β and LH-β subunits in the pejerrey fish, Odontesthes bonariensis

Fragments of the cDNAs encoding for FSH-β (428 bp) and LH-β (366 bp) subunits were studied in the pejerrey (pj), Odontesthes bonariensis, Pejerrey gonadotropins (GtHs) are close to percomorpha fish GtHs, LH-β has been better conserved than FSH-β during teleosts evolution.     

Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.     

Effects of sex and season in haematological parameters and cellular composition of spleen and head kidney of pejerrey (Odontesthes bonariensis)

Phylogenetic diversity in fish determines high interspecific variability in morphology as well as in physiological parameters. Moreover, several haematological variables and the organ composition of haemolymphopoietic sites may vary according to sex or season. The aim of this study was to establish the haematological parameters and the cellular composition of haemolymphopoietic organs in Odontesthes bonariensis, a commercially valuable fish species in Argentina, and also to determine gender or seasonal variations. Haematocrit exhibited the highest value in summer, while haemoglobin concentration was greater in summer and autumn. Erythrocyte count was higher in spring than autumn and winter, but did not differ with summer. The increase in these variables in seasons with higher water temperatures might be a compensatory mechanism to compensate the lower level of oxygen in the environment. Leucocyte formula and blast haemolymphopoietic cells in spleen and head kidney also showed annual variations since cells related to specific immune response, i.e., lymphocytes and thrombocytes, decrease in winter, whereas cells of the non-specific immune pathways, such as granulocyte, rise. The elevation of a particular type of circulating leucocyte was preceded by an increase in values of its precursor in blood in the previous season. Both, spleen and head kidney were active in haemolymphopoiesis, although with some differences in their activity during different seasons. Males showed higher values of circulating lymphoblasts and granulocytes than females, whereas females exhibited higher values of thrombocytes. This study corroborates the high interspecific variations in haematological parameters in fish that underlines the needing of basic studies in order to assess fish health status in new promising species for aquaculture.     

Neuroanatomical distribution and partial sequence of cDNA encoding brain cytochrome P450 aromatase (P450aromB) in pejerrey Odontesthes bonariensis

The molecular cloning of a cDNA encoding the pejerrey brain cytochrome P450 aromatase (P450aromB) is described. This form shares higher identity to other brain aromatases than with their respective ovarian counterparts and the self ovarian aromatase. Tissue-specific expression of both aromatases was examined in pejerrey by RT-PCR. The immunocytochemical distribution of P450aromB was described.     

Influence of pejerrey Odontesthes bonariensis (Valenciennes, 1835) broodstock age on gamete quality,reproductive performance and plasma sex steroid levels during the spawning season

This study evaluated the effect of pejerrey Odontesthes bonariensis (Valenciennes, 1835) broodstock age on gamete and spawning quality and its relationship with sex steroid plasma levels. Sperm was analysed using a computer‐assisted sperm analysis (CASA) developed for pejerrey. Semen samples were taken from all fish from mid age groups (5‐ and 7‐year fish), but it was not possible to obtain stripped semen samples from all fish of younger (3‐year) or older (10‐year) groups. The highest relative sperm weight and sperm concentration were recorded in 5‐ and 7‐year groups respectively, while viability was not different between age groups. It was not possible to identify an effect of age on sperm quality by CASA. Non‐viable spawned eggs were obtained in the 3‐year group and, the relative fecundity and the relative number of fertilized eggs decreased with age in other groups. Fertilization and hatching rates showed the highest values in the 10‐year group. However, the estimated number of hatched larvae was similar in 5‐, 7‐ and 10‐year fish. A clear effect of age on 11 ketotestosterone (11‐KT) and estradiol (E₂) levels was identified, with the highest values in the 5‐ and 7‐year groups. These results might be related to the onset of puberty in the 3‐year group and ageing in the 10‐year group. Considering that the estimated number of larvae obtained was similar among age groups, the results of this study suggest that broodstock maintenance cost could be reduced by using mid‐age fish rather than older fish in pejerrey hatcheries.     

The GnRH systems in the brain and pituitary of normal and hCG treated European silver eels

The distribution of immunoreactive GnRH was studied in the brain and pituitary gland of normal and human chorionic gonadotrophin (hCG) injected silver eels. It was found that the general organization of GnRH systems in this species is similar to that reported in other teleosts. Cell bodies were present in the olfactory bulbs, ventral telencephalon, periventricular hypothalamus and dorsal tegmentum. No positive perikarya could be detected in the preoptic region. Only scarce fibers were observed in the proximal neurohypophysis. Treatment with hCG does not modify the distribution of GnRH but it increases the density of positive structures, in particular at the level of the pituitary. The results are discussed in relation with the present status of knowledge of the mechanisms underlying the blockage of sexual maturation in the European eel at the silver stage.     

Effects of dietary sunflower oil on growth parameters,fatty acid profiles and expression of genes regulating growth and metabolism in the pejerrey (Odontesthes bonariensis) fry

Aquaculture fish diets usually contain an addition of fish oil to improve their nutritional value. The effect of the replacement of dietary fish oil (FO) by sunflower oil (SfO) on growth, fatty acid composition and expression of genes implicated in somatic growth, feed intake and fatty acid metabolism was studied in pejerrey fry. Fry were fed per 45 days with diets containing FO/SfO ratios of 100% FO; 50% FO:50% SfO; 20% FO:80% SfO; and 100% SfO. No differences were detected in growth and in the total per cent of saturated and monounsaturated fatty acids. Gh, ghr‐I and ghr‐II showed a higher mRNA expression in head and trunk of fry fed with 100% SfO diet. Expression of igf‐II was higher in trunk of fry fed with 100% SfO diet compared with 100% FO diet. The Δ6‐desaturase gene expression was upregulated in head and trunk of fry fed with 100% SfO diet. The nucb2/nesfatin‐1 gene expression decreased in the trunk of fry with increasing dietary SfO. We conclude that the replacement of fish oil by sunflower oil in pejerrey fry feed does not affect growth and is a viable strategy to reduce production costs of this fish.     

Temperature effects on sex differentiation of the reciprocal hybrids of Odontesthes bonariensis and Odontesthes hatcheri (Atherinopsidae)

The pejerrey Odontesthes bonariensis (Valenciennes 1835) and the Patagonian pejerrey Odontesthes hatcheri (Eigenmann 1909) are Atherinopsid species with commercial importance and potential for aquaculture. The hybrids of the two species are viable but their mode of sex determination is unknown. This study examined the gonadal histology and sex ratios of reciprocal hybrids that were reared at 15, 17, 21, 25 or 29 °C during the sex differentiation period. The genetic sex of hybrids from O. hatcheri fathers was inferred from a sex‐linked SNP marker. Both hybrids showed female‐biased sex ratios at the lowest temperature, female‐biased to balanced sex ratios at intermediate temperatures and balanced or male‐biased sex ratios at 29 °C, but unlike in purebred O. bonariensis, the lowest and highest temperatures did not yield monosex populations. The proportion of females in the offspring was affected more by parental genome than by hybrid combination. Female hybrids bearing the O. hatcheri Y chromosome showed temporary arrest of ovarian development that was rescued in adults. These results reveal strong interactions between genotype and temperature for sex determination and differentiation of the hybrids and provide important clues to understand the mechanisms of sex determination in these species.