Gonadotropin-releasing hormone (GnRH) neuronal systems in the pejerrey, Odontesthes bonariensis (Atheriniformes)

The brain of the pejerrey (Odontesthes bonariensis) has recently been shown to contain three forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and pejerrey GnRH (pjGnRH), nevertheless neuroanatomical studies on the distribution of these peptides are lacking. In this study we investigated the distribution of immunoreactive GnRH in the brain of adult pejerrey. Four different policlonal antisera and a monoclonal antibody against different GnRH variants were applied on cryosections and visualized using the ABC method. Three antisera (PBL#49, sGnRH#2 and cII741) revealed three different immunoreactive areas: the terminal nerve ganglion (at the junction between the olfactory bulbs and the anterior telencephalon), the preoptic area just anterior to the hypothalamus and the midbrain tegmentum. Fibers immunoreactive to GnRH were detected in different brain areas: the olfactory bulbs, the ventral thelencephalon, the hypothalamus, the mesencephalic area and an important innervation entering into the pituitary gland. Two other antibodies (LRH13 and s1668) labeled the two nuclei corresponding to the forebrain but not the midbrain tegmentum. As both antibodies have low crossreactivity to cGnRH-II, the data suggest that this group of cells express cGnRH-II. In summary, three different areas with immunoreactivity to GnRH were detected in the pejerrey brain. The distribution of sGnRH, pjGnRH and cGnRH-II expressing neurons, is discussed.     

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.     

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.     

Growth and feeding of Patagonian pejerrey Odontesthes hatcheri reared in net cages

An experiment was performed to evaluate the performance of Patagonian pejerrey during net cage rearing in the oligomesotrophic reservoir Exequiel Ramos Mexía. Survival, growth, nutrition and reproduction were evaluated for two lots of pejerrey, initial weights 4.0 and 2.5 g, reared in net cages for 22 and 14 months respectively. Fish were stocked at 29 and 48 individuals m^?3 densities and fed with an experimental pejerrey feed. During the experiment, temperature fluctuated between 6.6 and 19.6 °C. The two lots did not show substantial differences in survival (pooled survival at the end of the experiment >80%) and the thermal‐unit growth coefficient (TGC), daily feed intake and feeding efficiency were 0.43±0.19, 1.70±0.80 and 53.6±9.9 respectively. Digestive tract analysis showed that caged pejerrey can consume substantial quantities of natural food, taking advantage of its planktivorous condition. Pejerrey showed high percentage survival, slow growth and early sexual maturation in captivity. The use of the TGC is proposed as a model for describing the growth pattern of this species and other pejerrey under culture conditions.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Influence of the length of time after hormonal stimulation on selected parameters of milt of ide Leuciscus idus L.

Milt of the Leuciscus idus L. was collected from five experimental groups, and selected parameters of its quality were analysed for 36 h (group II), 60 h (group III), 84 h (group IV) and 108 h (group V), respectively, after hormonal stimulation with Ovopel (1 granule kg^?1 of body weight). The control (group I) fish were not subjected to hormonal stimulation. The highest milt volume was obtained from the fish in group IV (0.70 ± 0.55 mL), where the largest volume of milt expressed per kilogram was also obtained (3.03 ± 1.94 mL kg^?1). Significant differences were also found in milt volumes obtained between group I and groups III (P<0.01) and IV (P<0.05). The highest percentage of motile spermatozoa was found in the milt of group IV (59%); significant differences were found between group I and groups II (P<0.01) and III (P<0.001). The value of osmotic pressure of seminal plasma was the highest in group IV (203.19 ± 37.63 mOsm kg^?1), and the lowest in group I (118.31 ± 41.13 mOsm kg^?1). Parameters determining milt quality and quantity indicate that the period of 60–84 h after hormonal stimulation with Ovopel is optimal for obtaining milt from ide.     

Spontaneous hybridization in the laboratory and genetic markers for the identification of hybrids between two atherinid species, Odontesthes bonariensis (Valenciennes, 1835) and Patagonina hatcheri (Eigenmann, 1909)

This study reports the occurrence of spontaneous hybridization between Odontesthes bonariensis (Valenciennes, 1835) and Patagonina hatcheri (Eigenmann, 1909), two South American freshwater atherinids, while adults of both species were being held in a communal laboratory tank, and describes suitable genetic markers for distinction of both species and their hybrids. Combined allozyme and mitochondrial DNA restriction length polymorphism analyses of offspring from three spawnings in the communal tanks confirmed the presence of hybrids along with purebreds in each spawning. Hybrids of an O. bonariensis mother X a P. hatcheri father were produced along purebred O. bonariensis in one occasion, whereas hybrids of P. hatcheri mothers X O. bonariensis fathers were produced along purebred P. hatcheri in two occasions. Three enzyme systems (coded by loci LDH-B*, PGM*, and CK*) out of 15 (20 loci) screened allowed distinction of species and their hybrids, but the usefulness of LDH-B* for field work may be limited by intraspecific polymorphism. Eight restriction enzymes (Apa I, Bgl II, Eco RI, Eco RV, Eco T22I, Hinc II, Hind III, and Pvu II) among 11 produced diagnostic restriction fragments applicable for species (motherhood) distinction. The relevance of these findings for the management of natural genetic resources and the aquaculture of these two species is discussed.     

Influence of gonadotropic hormone-releasing hormone analog (GnRH-A) on plasma sex steroid profiles and milt production in male winter flounder,Pseudopleuronectes americanus (Walbaum)

The effects of gonadotropic hormone-releasing hormone analog (GnRH-A) treatment on the onset and duration of increases in plasma sex steroids and milt production (milt volume and number of spermatozoa) were investigated in prespawning male winter flounder. After treatment of maturing males during the winter with a single injection of either 20 or 200 g/kg [D-Ala⁶, Pro⁹-NHEt]LHRH (GnRH-A), plasma levels of testosterone and 11-ketotestosterone were increased within 12h and the steroid hormone levels remained elevated for long periods lasting several days. The androgenic steroid response of males was delayed after the administration of a lower dose of GnRH-A (2 g/kg). Although a single GnRH-A injection in December or January advanced the onset of spermiation in some males, only small amounts (<50 l) of milt could be collected. By March, all males were in spermiating condition following GnRH-A treatment; however, significant increases in sperm production, particularly increases in milt volume, occurred in fish twice treated with GnRH-A.Part of this work presented at the First Symposium of Fish Endocrinology, Edmonton, Alberta, Canada June 1988 and the Satellite Symposium on Applications of Comparative Endocrinology to Fish Culture, Almuñecar, Granada, Spain May 1989     

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.     

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.     

Gonadotropin releasing hormone‐analogue (GnRHa) treatment improves the milt production and sperm motility of endangered Caspian brown trout,Salmo trutta caspius,over the course of a spawning season

To determine the effect of gonadotropin releasing hormone‐analogue (GnRHa) treatment on the milt quality of endangered Caspian brown trout, Salmo trutta caspius, the sperm motility (percentage and duration of motility), sperm production (sperm density, spermatocrit and milt volume) and milt pH were measured for GnRHa‐treated (the treatment group) and untreated groups (the control group) during the spawning season. For untreated brooders, the values of the motility per cent, sperm density and spermatocrit decreased continuously during the spawning season while the milt volume, duration of motility and milt pH showed only a significant decrease at the end of the season. For GnRHa‐treated males, these parameters increased 14 days after GnRHa treatment (first milt collection) and then decreased continuously towards the end of the season. In addition, the values of milt and sperm density yielded per treated male were higher than that in the untreated group, although these were not statistically different. In any case, the total sum of yielded milt from the treatment group over the spawning season was higher than that in the untreated group. In this experiment, significant positive correlations were found between milt parameters as follows: sperm motility vs. milt pH; sperm density vs. spermatocrit; milt volume vs. spermatocrit; and milt volume vs. sperm density. The results show that the treatment of Caspian brown trout by GnRHa can improve the milt quality in terms of sperm motility and sperm production during a spawning season.     

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.     

Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus)

In two separate spawning seasons, spermiating male Atlantic halibut were implanted with pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Males were bled repeatedly, and milt samples were collected. Blood samples were assayed for free and conjugated steroids: testosterone, 11-ketotestosterone, 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20,21-trihydroxy-4-pregnen-3-one and steroids with a 17,20 configuration. Towards the end of the first season, pellets were implanted into three wild-caught and three hatchery-reared males. No control fish were available. The major progestogen in plasma was identified as sulphated 5-pregnane-3,17,20-triol (3,17,20-P-5-S). Concentrations of this steroid were stimulated by the GnRHa. Sulphated 17,20-P was also identified in the plasma, but at 10-fold lower concentrations than 3,17,20-P-5-S. In the middle of the second season, pellets were implanted into five hatchery-reared males; five unimplanted males were used as controls. Levels of androgens fell following GnRHa treatment, levels of progestogens rose briefly, and there was a significant increase in the fluidity of the milt. Of all the measured steroids, free and sulphated 17,20-P showed the best correlation with milt fluidity.