Pejerrey Fish Research Articles

Effects of Anthropic Pollutants Identified in Pampas Lakes on the Development and Reproduction of Pejerrey Fish Odontesthes bonariensis

Anthropic activities can seriously affect the health of the organisms inhabiting them, and the observation of any alteration in the reproduction of fish could be associated with the presence of endocrine disruptors. In this manuscript we have collected information on the adverse effects of pollutants (heavy metals, environmental steroids, and agrochemicals), present in Chascomús lake, Argentina, either at environmentally relevant and pharmacological concentrations on reproduction, embryonic development, and larval survival of pejerrey fish Odontesthes bonariensis. During development, it has been reported that 17β-estradiol (E2) feminized and reduced larval survival, while 17α-ethinyl-estradiol (EE2) not only feminized but also affected both embryo and larval survival. In adult male fish, treatments with EE2 and E2 + EE2 were able to increase mRNA abundance of gnrh3 and cyp19a1b and decreased those of gonadotropin receptors (fshr and lhcgr). Heavy metals such as cadmium, chromium, and copper negatively affected sperm quality, diminishing the motility. Also, a decrease in the percentage of hatching rate and larval survival was also observed with the same metals, highlighting zinc as the most detrimental metal. Furthermore, all these metals altered the expression of hypothalamic and pituitary genes related to reproduction in male pejerrey (gnrh1,2,3; cyp19a1b; fshb; lhb; fshr and, lhcgr). Moreover, in all cases pyknotic cells, corresponding to the degeneration of the germ cells, were observed in the testes of exposed fish. For agrochemicals, exposure of male pejerrey to environmental concentrations of glyphosate did not cause alterations on the endocrine reproductive axis. However, male pejerrey with gonadal abnormalities such as the presence of intersex (testis-ova) gonads were found in other Pampa´s lakes with high concentrations of atrazine and glyphosate associated with soybean and corn crops near their coasts. These types of studies demonstrate that pejerrey, an endemic species with economic importance inhabiting the Pampas shallow lakes, can be used as a sentinel species. It should be noted that increased pollution of aquatic ecosystems and the effects on the reproduction of organisms can lead to a decline in fish populations worldwide. Which, added to overfishing and other external factors such as global warming, could cause an eventual extinction of an emblematic species.

Gonadal development in a landlocked population of pejerrey fish Odontesthes argentinensis during spawning season in relation to sex steroids and water temperature

ABSTRACT The relationship between water temperature and gonadal maturation of a landlocked ecophenotype of marine pejerrey (Odontesthes argentinensis) in La Salada de Pedro Luro lake was analysed during three spawning seasons. Mainly vitellogenic females were found in August (winter), some ovulating in October (mid-spring) and with atretic oocytes in December (end of spring). In accordance with this, gonadosomatic index (GSI) and estradiol plasma levels showed maximum values in October (7.6 ± 0.7%; 1631.7 ± 256.1 pg/mL) and minimum values in December (1.2 ± 0.3%; 230,33 ± 8,74 pg/mL). Spermiating males were found during the whole spawning season and only arrested in December. The highest GSI values were recorded in October (4.6 ± 0.2%) and for testosterone (T) in August (1113.2 ± 503.8 pg/mL). Meanwhile, in December the minimum GSI and T plasma values (1.3 ± 0.6%, 323.8 ± 5.3 pg/mL) were observed. According to the Fourier fitted models, temperature explains more than 70% of the observed variation in GSI in both sexes. Moreover, the total GSI variation range was positively related to the total temperature range variation experienced during the reproductive seasons demonstrating the influence of this environmental variable on maturation intensity and dynamics.

Comparative Analysis of Pejerrey Fish (Odontesthes bonariensis) Gonadal Development During Two Consecutive Spawning Seasons in Relation to Sex Steroids and Temperature Variation in Cochicó Lake (Pampas Region, Argentina)

Cochicó belongs to “Encadenadas del Oeste” system of lakes being a typical water body of the Pampas region. The most abundant fish species in this lake is the pejerrey (Odontesthes bonariensis) valued due to the quality of its flesh and as a game fish. The aim of this study was to compare the gonadal stages of pejerrey during two consecutive spawning seasons (August to December) in relation to sexual steroids and temperature in this lake. In general, pejerrey gonadal development, the Gonadosomatic index and the plasma levels of estradiol and testosterone fluctuated in relation with temperature. In 2014 samplings, females started to ovulating in early August, with a peak during September-October and ending in December with many of them with atretic oocytes. However, in 2015, a marked delay in maturation was observed with ovulated fish only in October and December. This fact may be because the minors mean temperatures recorded in this year. For males, it was possible to find spermiating animals during the whole spawning season and only arrested animals in December. Unexpectedly, histological gonadal analysis revealed for the first time pejerrey with testis-ova, probably due to the intensive use of agrochemicals in this region.

Effects on Ultrastructure, Composition and Specific Surface Area of the Gills of Odontesthes bonariensisUnder Subchronic Glyphosate-Based Herbicide Exposure.

Gills represent one of the major sites of gas exchange of fish, consequently they are in continuous close contact with the aquatic environment and its pollutants. In the present study the effects on gills of pejerrey fish, Odontesthes bonariensis, under glyphosate-based herbicide subchronic exposure were analyzed. Adult animals were exposed to sublethal concentrations of a glyphosate-based commercial formulation (1 and 10 PMG mg L-1, PMG: glyphosate active ingredient) for 15 days, while control group was maintained in rearing water. Ultrastructural changes in gills were observed by scanning electron microscopy (SEM). The composition of the surface epithelium and specific surface area were determined by energy dispersive spectroscopy (EDS) and N2 (g) adsorption-desorption isotherms, respectively. The herbicide exposure induced severe alterations in gill ultrastructure, as shown in the SEM micrographs. Accordingly, an increase in surface area of the gills of exposed animals was determined. These results support that gills parameters of freshwater fish are sensitive morphological biomarkers for glyphosate exposure.

Effects of metals on sperm quality, fertilization and hatching rates, and embryo and larval survival of pejerrey fish (Odontesthes bonariensis).

Some species of fish have been used as bioindicators of aquatic environmental pollution all over the world. Pejerrey (Odontesthes bonariensis) was selected for the current study due to its sensitivity to pollutants and because is one of the emblematic fish species that inhabits shallow lakes of the Pampa region (Argentina). Recently, in Chascomús lake were recorded concentrations of Cd, Cr, Cu and Zn with values above the Argentine National Guidelines for the Protection of the Aquatic life. Regarding this, the aim of the present study was to investigate the effects of environmental concentrations of these metals on the sperm quality, fertilization and hatching rates, and embryo and larval survival of pejerrey. Also, the same endpoints were analyzed with concentrations ten times higher to simulate a polluted worst-case scenario. The results showed that the presence of some metals in aquatic environments reduced pejerrey sperm motility (in ~50%) and velocity (in ~30%). These results were obtained using a computer assisted sperm analyzer enforcing the application of this analysis as a tool or bioindicator of aquatic pollution. In addition, fertilization rate was diminished (in ~40%) for all treatments. Besides, the hatching rate, and embryo and larval survival were drastically affected being zero for the highest metal concentrations assessed. All together these results, showed that even lower metal concentrations can negatively affect different reproductive parameters of one of the most emblematic fish species of the Argentinean water bodies.

Cooling of pejerrey Odontesthes bonariensis (Teleostei, Atherinidae) embryos at sub-zero temperatures

Pejerrey fish (Odontesthes bonariensis) is a seasonal multiple spawner with great economic importance and an adequate species for Aquaculture. For these reasons, it is necessary to apply biotechnologies to optimize its reproduction in captivity. In this context, the aim of this work was to develop a cooling protocol for pejerrey embryos at sub-zero temperatures. Two cryoprotective solutions (CSs: S1 and S2), two cooling curves (a fast and a slow one) and two storage temperatures (−14 and −20 °C) were evaluated for 1 h. High percentages of embryo survival (80–100%) were obtained in all cases. In particular, for cooling at −14 °C, the most suitable protocol was the slow temperature decrease in combination with S1 (2.5 M methanol, 1.4 M Me2SO, 0.3 M sucrose, and 0.08 M NaCl). The hatching rate (86.67 ± 11.55%) and the larval survival observed did not differ from those of the control group, and about 30% of normal-looking larvae were obtained. Besides, the slow cooling was also the best way to reach −20 °C, obtaining a hatching rate of around 60%. However, all the larvae had different kind of malformations. Finally, in order to improve the results obtained at −20 °C, the CSs were incorporated into the embryos by microinjection. In this case, it was observed that the most convenient combination was the microinjection of S2 (same composition as S1 but without Me2SO) in the perivitelline space followed by rapid cooling. Although the hatching rate was not improved (67.93 ± 8.31%), the microinjection allowed to obtain at least 4.5% normal-looking larvae. These results showed that the cooling of pejerrey embryos at zub-zero temperatures was feasible. Moreover, the microinjection of cryoprotectants within the pejerrey O.bonariensis embryos was employed for the first time in this species.

Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish.

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey.

Effects of heavy metals identified in Chascomús shallow lake on the endocrine-reproductive axis of pejerrey fish (Odontesthes bonariensis)

Some heavy metals related to human activities were measured in the water of Chascomús lake. The maximum concentrations were: 0.23 μg/L for Cd, 4.28 μg/L for Cr, 22.09 μg/L for Cu, 2.49 μg/L for Ni, 3.24 μg/L for Pb and 210.76 μg/L for Zn. The values of Cd, Cr, Cr, Pb and Zn were above the Argentine National Guidelines for the Protection of the Aquatic life. The analysis of gonadal condition of pejerrey fish (Odontesthes bonariensis) from this lake did not revealed any reproductive damages. However, exposures with environmental concentrations of Cd, Cr, Cu and Zn under laboratory conditions of pejerrey males (14 days), caused a significant increase of the expression of the three variants of gnrh in the brain (within Cd exposure) and a decrease in cyp19a1b mRNA (within Cu exposure). Furthermore, at pituitary level, a decrease in fshb transcript levels was observed in the fish exposed to Cd and Cr and a decrease in the expression of both gonadotropin receptors at gonadal level in Zn exposure. Moreover, the gonads of the fish exposed to all the tested metals suffered structural damages showing shortness of the spermatic lobules, fibrosis, testis ova and the presence of piknotic cells. All these findings alert that heavy metals pollution affects the expression of key reproductive genes and gonadal structure of fish species that represent the predominant group of organisms and are considered sentinel species in the aquatic ecosystems.

Exposure to E2 and EE2 environmental concentrations affect different components of the Brain-Pituitary-Gonadal axis in pejerrey fish (Odontesthes bonariensis)

The present study focuses on the effects of E2 and EE2 environmental concentrations on different components of the reproductive axis of pejerrey (Odontesthes bonariensis), a native fish species from Pampas lakes of Argentina. The results obtained demonstrated that E2 and EE2 separate or mixed, could disrupt key pathways of the pejerrey Brain-Pituitary-Gonadal axis. First, it was observed that at the brain level, gnrh-III and cyp19a1b mRNA expression increased significantly in the exposed fish. Secondly, in the pituitary fshb and lhb mRNA expression levels, the study did not show any differences between treated and control groups. Thirdly, fshr and lhcgr transcript levels showed a significant decrease at testicular level. Nevertheless, testosterone plasmatic levels remained unchanged in exposed fish. In addition, in a histological analysis, it was possible to find pyknotic nuclei in estrogen only on treated fish testis linked to a reduction in the GSI index and a decrease in the length of spermatogenic lobules. All these findings highlighted the fact that environmental concentrations of E2, EE2 and their mixture disrupted the endocrine-reproductive axis of pejerrey, being the testis the main direct target.

Sex hormone binding globulin: Expression throughout early development and adult pejerrey fish, Odontesthes bonariensis

Sex hormone binding globulin (Shbg) is a plasma glycoprotein that binds and transports steroids in the blood of all vertebrate classes apart from birds. In the present study we characterized shbg from pejerrey, a fish species with a well characterized temperature-dependent sex determination. The pejerrey shbg mRNA comprises 1185bp encoding for a 395 amino acid Shbg precursor protein that includes a leader sequence for secretion. Relative quantification of shbg transcript abundance revealed expression early in development coinciding with the sex-determining period and probably in association with temperature leading to male determination. The hepatopancreas was the main site of shbg expression, which varied according to the sex cycle in females. It was also expressed in gills, gonads, gut and taste buds during both larval stages and in adult fish. The presence of Shbg in organs in close contact with the environment such as gills, pseudobranchs, gut and taste buds suggests that these are potential sources of uptake or release of steroids/xenosteroids to and from the aquatic environment.

Kisspeptin system in pejerrey fish (Odontesthes bonariensis). Characterization and gene expression pattern during early developmental stages

In vertebrates, kisspeptins and their receptors are known to be related to puberty onset and gonadal maturation, however, there are few studies concerning their role in early development. Here, we characterize the kisspeptin system in the pejerrey, Odontesthes bonariensis, a fish with strong temperature-dependent sex determination. We reconstructed the phylogenetic history of the two ligands (kiss1 and kiss 2) and two receptors (kissr2 and kissr3) in pejerrey in the context of recent classifications of bony fishes, determined their tissue distribution and documented the early expression pattern of these ligands and receptors. Phylogenetic analysis of these gene families clearly resolved the percomorph clade and grouped pejerrey with Beloniformes. Paralogous sets of genes putatively arising from the teleost-specific genome duplication event (3R) were not detected. Kisspeptins and their receptors showed a wide tissue distribution in adult pejerrey, including tissues not related to reproduction. In larvae reared at 24°C, the four kisspeptin elements were expressed in the head from week 1 to week 8 of life, with no differences in transcript levels. Larvae kept at a female-producing temperature (17°C) did not show statistically significant differences in the transcript levels of all analyzed genes during the sex determination/differentiation period; however, in those larvae raised at male producing temperature (29°C), kiss2 levels were increased at week 4 after hatching. These results showed that all members of the kisspeptin system are expressed at this early period, and the increase of kiss2 transcripts at week 4 could be interpreted as it would be related to the differentiation of the brain-pituitary axis in male development.

Effects of estradiol and ethinylestradiol on sperm quality, fertilization, and embryo–larval survival of pejerrey fish (Odontesthes bonariensis)

17β-Estradiol (E2) and synthetic 17α-Ethinylestradiol (EE2) are estrogenic compounds present in surface waters as a consequence of municipal sewage discharges. The aim of this study was to evaluate the effects of E2, EE2 and its mixtures on different reproductive parameters and embryo–larval survival in pejerrey fish (Odontesthes bonariensis). In order to analyze the effect of these compounds on sperm quality, fertilization%, embryo–larval survival (%), and the point of no return (PNR), different assays were performed using concentrations 175, 350, 700 and 1400ng/L of E2; 22.5, 45, 90 and 180ng/L of EE2 and mixtures M1 (175 E2+22.5 EE2, ng/L), M2 (350 E2+45 EE2, ng/L), M3 (700 E2+90 EE2, ng/L) and M4 (1400 E2+180 EE2 ng/L). No significant differences in motility parameters were observed between E2 and EE2 treatments and the control group. However, a significant decrease in motility% was recorded for all mixtures tested compared with the control samples. For fertilization%, only sperm activated with M4 showed a significant decrease compared with the control group. In the case of embryo survival, there was only a significant decrease in the highest concentration of EE2 compared with the control group. For the mixtures, M3 is the one that had the most adverse effect on embryo survival. In larval survival, there was a significant decrease in concentration 175 and 700ng/L of E2 compared with the control group. In EE2 treatments, the ones with a significant reduction in larval survival were concentration 45 and 90ng/L. And for the mixture treatments, M1, M3 and M4 had a significantly lower larval survival than the control group. In comparison to other treatments, M1 demonstrated a significant difference in PNR when compared with the control group. The results obtained demonstrated that the exposure to mixtures of E2 and EE2 affected fish sperm motility, fertilization% and, embryo and larval survival even at relevant environmental concentrations highlighting the necessity of considering the effects of pollutants mixtures in ecotoxicological studies.

Dietary phosphorus requirement of pejerrey fingerlings (Odontesthes bonariensis)

To determine the available phosphorus requirement of pejerrey fish, four semi-purified diets were formulated to contain increasing available phosphorus levels (0.90, 2.7, 5.7 and 8.3 g/kg). Each diet was assigned to groups of 10 fish with average weight of 1.26±0.17 g, distributed into a completely randomized design with four treatments and four replications. Fish were fed at a rate of 10% total biomass four times a day for 60 days. The parameters productive performance, body chemical composition and mineral composition of bones and scales were evaluated. The best productive performance of fingerlings was obtained with the diet containing 4.3 g/kg available phosphorus. The average level of 6.3 g/kg available phosphorus promotes better mineralization of bones and scales in pejerrey fish.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Brain aromatase (Cyp19A2) and estrogen receptors, in larvae and adult pejerrey fish Odontesthes bonariensis: Neuroanatomical and functional relations

Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, α and β, in pejerrey. Both receptors’ mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males. In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors’ mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor α, but not β, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase. Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature. Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate.

6.P1. Identification of different population of neural progenitor in adult pejerrey fish: Origin of proliferation, migration and cell fate

6.P1. Identification of different population of neural progenitor in adult pejerrey fish: Origin of proliferation, migration and cell fate

Characterization of the cDNAs Encoding Three GnRH Forms in the Pejerrey Fish Odontesthes bonariensis (Atheriniformes) and the Evolution of GnRH Precursors

Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main paralogous GnRH lineages with different function. A "forebrain lineage" separates evolutionarily from the "midbrain lineage" as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from chromosomal studies of synteny around the GnRH genes in vertebrates.

Dmrt1 expression analysis during spermatogenesis in pejerrey, Odontesthes bonariensis

Dmrt1, a putative transcription factor with a DM-binding motif (called the DM domain), is generally considered to be exclusively expressed in testicular tissue, except in some teleost species where it has also been demonstrated to be expressed at the ovary. The aim of the present manuscript was to characterize the dmrt1 cDNA and analyze its expression pattern at different spermatogenetic stages in the pejerrey fish, Odontesthes bonariensis. The pejerrey dmrt1 (pjdmrt1), encodes for a 290 amino acid protein including the conserved DM and P/S domains. The sequence comparison of the pjdmrt1 revealed a high level of identity with different dmrt1 from other vertebrates and the phylogenetic analysis showed the existence of two main clades: one represented by the mammalian dmrt1 sequences and the second by dmrt1 sequences from teleost fishes. This pattern may represent the evolution of this molecule. In pejerrey fish, the expression of this gene was exclusively restricted to the testis at all spermatogenetic stages analyzed, and pjdmrt1 expression levels varied according spermatogenetic stages suggesting a role for dmrt1 in the adult testicular function.

Molecular cloning, expression and immunological characterization of pejerrey (Odontesthes bonariensis) growth hormone

Growth hormone is an essential polypeptide required for normal growth and development of vertebrates. The pejerrey fish, Odontesthes bonariensis, is a South American atherinid freshwater fish considered as a promising species for aquaculture. Although growth hormone has been characterized in a number of fish, there are no published data on the structure of this hormone in atherinids, except that of a related species Odontesthes argentinensis. In this paper, the molecular cloning, expression and immunological characterization of pejerrey growth hormone (pjGH) is described. The predicted amino acid sequence of pjGH cDNA consisted of 204 amino acid residues with an estimated molecular mass of 23 kDa. Amino acid sequence was highly conserved among the two Atheriniformes where the growth hormone sequences are known (99% aa identity), highly to moderately conserve (75–92% aa identity) when compared to the other members of Acantopterigii superorder and clearly less conserved (49–66% identity) when compared to Salmoniformes (Protacanthopterygii), Cypriniformes and Siluriformes (Ostariophysi). A phylogenetic tree depicting the relationship of various teleost GH nucleotide sequences was inferred. Pejerrey GH was produced using recombinant DNA technology in a bacterial system, representing the first time an atherinid growth hormone protein was expressed as a recombinant protein in Escherichia coli. A specific antiserum of this hormone was raised in rabbits and its specificity tested by using Western blot and immunocytochemistry. The distribution of pjGH mRNA was also studied by RT-PCR and Southern blot analysis. The transcript was detected not only in the pituitary gland but also in the testis.

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 ¢sh 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 ¢sh, is a major problem. The aim of this work was to study the eiectiveness of the administration of diierent 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 (12.8fold increase, 30 mg kg 1 ), salmon-type gonadotropin-releasing hormone analogue (GnRH) (16.7-fold increase, 10m gk g 1 ) and mammalian-type GnRH analogue (10.8-fold increase, 20m gk g 1 ). Sperm concentration, motility and the fertilization rate were not statistically diierent compared with control groups. It was also demonstrated that sperm could be obtained oi-season. Taken together, hCG is recommended to stimulate pejerrey spermiation because it is eiective in low doses is inexpensive and is widely available.