Spectrophotometric method for determination of Colossoma macropomum (tambaqui) sperm concentration / Determinação da concentração espermática de tambaqui (Colossoma macropomum) por espectrofotometria

To assist the reproductive management of tambaqui (Colossoma macropomum) males in laboratory and commercial fish farming, a linear regression model was obtained from concentration curves using the spectrophotometric method. Twenty-two tambaqui males with an average age of three years old were selected and divided into two groups containing 11 animals each. Both groups alternately received a single dose of carp pituitary extract (CPE; 2.0 mg/kg body weight, intracoelomic). Sperm was collected 14 h after hormonal treatment and diluted (1:4000; sperm:formaldehyde saline). The concentration was estimated by counting spermatozoa in a Neubauer chamber and by using a spectrophotometer (λ=540 nm). Individual sperm concentration ranged from 11.40 to 71.13 × 109 sperm/mL. The degree of transmittance ranged from 62.1% to 95.0%. There was a significant correlation (r2 = 0.966; p < 0.0001) between sperm concentration analyzed in a Neubauer chamber and transmittance at 540 nm. Analysis by spectrophotometry and the prediction provided by the equation Y=100.293 - 0.509X proved to be an efficient and fast method for estimating sperm concentration in tambaqui and can be used in routine procedures in artificial fish reproduction laboratories.

Visando auxiliar o manejo reprodutivo de machos de tambaqui (Colossoma macropomum) em piscicultura de laboratório e comercial, obteve-se um modelo de regressão linear a partir de curvas de concentração por método espectrofotométrico. Foram selecionados 22 machos de tambaqui com idade média de três anos. Eles foram divididos em dois grupos contendo 11 animais cada. Ambos os grupos receberam alternadamente uma única dose de extrato de hipófise de carpa (EHC; 2,0 mg/kg de peso corporal, intracelomático). O esperma foi coletado 14 horas após o tratamento hormonal e diluído (1:4000; esperma: solução salina formaldeído). A concentração foi estimada por contagem de espermatozoides em câmara de Neubauer e por espectrofotômetro (λ=540 nm). A concentração espermática individual variou de 11,40 a 71,13 × 109 espermatozoides/mL. O grau de transmitância variou de 62,1 a 95,0%. Houve correlação significativa (r2 = 0,966; p < 0,0001) entre a concentração espermática analisada em câmara de Neubauer e a transmitância em 540 nm. A análise por espectrofotometria e a predição pela equação Y=100,293 - 0,509X mostrou ser um método eficiente e rápido para estimar a concentração espermática de tambaqui, podendo ser utilizado em procedimentos de rotina em laboratórios de reprodução artificial de peixes.

Dimethyl acetamide and dimethyl sulfoxide associated at glucose and egg yolk for cryopreservation of Pseudoplatystoma corruscans semen

This study aimed to develop a protocol for the cryopreservation of Pseudoplatystoma corruscans semen. For this, mature males were hormonally induced with a single dose of carp pituitary extract (5 mg/kg body weight). Semen was collected and evaluated. Two cryoprotectants were tested to compose the diluents: dimethyl acetamide (DMA) and dimethyl sulfoxide (Me2SO), in two concentrations (8% and 10%), + 5.0% glucose + 10% egg yolk. The semen was diluted in a 1: 4 ratio (semen: extender), packed in 0.5 mL straws and frozen in a dry shipper container in liquid nitrogen vapors. After thawing, sperm kinetics, sperm morphology and DNA integrity of cryopreserved sperm were evaluated. Pseudoplatystoma corruscans males produced semen with sperm motility > 80%. After thawing, all treatments provided semen with total sperm motility > 40%, with no significant difference (P < 0.05) between them, as well as between the other sperm kinetic parameters evaluated. The treatments with DMA provided a smaller fragmentation of the DNA of the gametes. Sperm malformations were identified in both fresh and cryopreserved semen, with a slight increase in these malformations being identified in sperm from thawed P. corruscans semen samples.(AU)

Este estudo teve como objetivo desenvolver um protocolo para a criopreservação do sêmen de Pseudoplatystoma corruscans. Para tal, machos maduros foram induzidos hormonalmente com uma dose única de extrato de hipófise de carpa (5 mg/kg de peso vivo). O sêmen foi coletado e avaliado. Sendo testados para compor os diluentes, dois crioprotetores: dimetil acetamida (DMA) e dimetil sulfóxido (Me2SO), em duas concentrações (8% e 10%), + 5,0% glicose + 10% gema de ovo. O sêmen foi diluído na proporção 1: 4 (sêmen: extensor), embalado em palhetas de 0,5 mL e congelado em container dryshipper em vapores de nitrogênio líquido. Após o descongelamento, foram avaliados os aspectos cinéticos espermáticos, a morfologia espermática e a integridade do DNA dos espermatozoides criopreservados. Os machos de P. corruscans produziram sêmen com motilidade espermática > 80%. Todos os tratamentos proporcionaram após o descongelamento sêmen com motilidade espermática total > 40%, sem diferença significativa (P < 0,05) entre eles, como também entre os demais parâmetros cinéticos espermáticos avaliados. Os tratamentos com DMA proporcionaram uma menor fragmentação do DNA dos gametas. Malformações espermáticas foram identificadas, tanto no sêmen fresco, como no criopreservado, sendo identificado um aumento discreto dessas malformações nos espermatozoides das amostras de sêmen descongeladas de P. corruscans.(AU)

Wild Leporinus friderici induced spawning with different dose of mGnRHa and metoclopramide or carp pituitary extract

Breeding technology is of utmost importance for reproduction of wild fish in captivity for the reintroduction and selective breeding programs purposes. The main challenge is that when applied to wild undomesticated specimens, conventional protocols often cause breeders and/or embryo mortality and spawning failure. In this study, we evaluated the reproductive performance of wild Leporinus friderici, a great importance fish for subsistence fishing in South American rivers, applying conventional and lower-dose hormonal therapies by means of two consecutive experiments. In the first, a conventional (0.5 and 5.5 mg/kg) and a lower carp pituitary extract (CPE) dose (0.5 and 1.0 mg/kg) were applied. In the second, a conventional mammalian GnRH analogue associated with metoclopramide (mGnRHa + MET) (40 µg mGnRHa + 20 mg MET/kg) and a lower dose (4 μg mGnRHa + 2 mg MET/kg and 8 µg + 4 mg of mGnRHa + MET/kg) were applied. Ovulation was observed in all treatments, however, only lower CPE protocol provided viable embryos. High levels of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17β estradiol (E2) detected in conventional, but not in lower CPE dose, at ovulation, might be associated to the mortality of the embryos. The use of lower CPE dose applied here was the best way to obtain L. friderici viable embryos. These results directly contribute to the knowledge about poorly explored effects of reproductive management and hormonal therapies in wild-type breeders, showing that the use of reduced doses may be an alternative to reproductive success.

Wild Leporinus friderici induced spawning with different dose of mGnRHa and metoclopramide or carp pituitary extract

Breeding technology is of utmost importance for reproduction of wild fish in captivity for the reintroduction and selective breeding programs purposes. The main challenge is that when applied to wild undomesticated specimens, conventional protocols often cause breeders and/or embryo mortality and spawning failure. In this study, we evaluated the reproductive performance of wild Leporinus friderici, a great importance fish for subsistence fishing in South American rivers, applying conventional and lower-dose hormonal therapies by means of two consecutive experiments. In the first, a conventional (0.5 and 5.5 mg/kg) and a lower carp pituitary extract (CPE) dose (0.5 and 1.0 mg/kg) were applied. In the second, a conventional mammalian GnRH analogue associated with metoclopramide (mGnRHa + MET) (40 µg mGnRHa + 20 mg MET/kg) and a lower dose (4 μg mGnRHa + 2 mg MET/kg and 8 µg + 4 mg of mGnRHa + MET/kg) were applied. Ovulation was observed in all treatments, however, only lower CPE protocol provided viable embryos. High levels of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17β estradiol (E2) detected in conventional, but not in lower CPE dose, at ovulation, might be associated to the mortality of the embryos. The use of lower CPE dose applied here was the best way to obtain L. friderici viable embryos. These results directly contribute to the knowledge about poorly explored effects of reproductive management and hormonal therapies in wild-type breeders, showing that the use of reduced doses may be an alternative to reproductive success.(AU)

Uso de gema de ovo em diferentes concentrações na congelação espermática de Prochilodus brevis e sua influência em parâmetros cinéticos / Use of egg yolk on Prochilodus brevis sperm freezing and its influence on kinect parameters

Prochilodus brevis is a fish species with an important ecological and economic role. Therefore, its captive breeding, together with sperm cryopreservation, come as an alternative to improve its availability. To perform sperm cryopreservation with quality results, it is necessary to develop studies on cryomedia composition, such as non-permeable cryoprotectants. The aim of the study was to test the efficacy of two non-permeable cryoprotectants on post-thawed kinetic parameters of Prochilodus brevis sperm. Twenty reproductive mature P. brevis males were selected and induced to sperm with carp pituitary extract. The sperm was collected, and five pools were made. Pools were diluted in 5% glucose and 10% DMSO added or not with three different concentrations of egg yolk (5; 10 or 12%). The samples were placed on French straws, frozen in dry shipper and stored in liquid nitrogen. After thawing, the kinetic analyses of computer assisted sperm analysis (CASA) were performed. There were no significant differences between the total motility of the control (60.48%) and treatments (49.0; 47.8 and 51.68%, respectively). The other kinetic parameters also showed no statistical difference. Thus, there is no need to use egg yolk in the freezing of P. brevis sperm when using a glucose and DMSO extender.

Uso de gema de ovo em diferentes concentrações na congelação espermática de Prochilodus brevis e sua influência em parâmetros cinéticos / Use of egg yolk on Prochilodus brevis sperm freezing and its influence on kinect parameters

Prochilodus brevis is a fish species with an important ecological and economic role. Therefore, its captive breeding, together with sperm cryopreservation, come as an alternative to improve its availability. To perform sperm cryopreservation with quality results, it is necessary to develop studies on cryomedia composition, such as non-permeable cryoprotectants. The aim of the study was to test the efficacy of two non-permeable cryoprotectants on post-thawed kinetic parameters of Prochilodus brevis sperm. Twenty reproductive mature P. brevis males were selected and induced to sperm with carp pituitary extract. The sperm was collected, and five pools were made. Pools were diluted in 5% glucose and 10% DMSO added or not with three different concentrations of egg yolk (5; 10 or 12%). The samples were placed on French straws, frozen in dry shipper and stored in liquid nitrogen. After thawing, the kinetic analyses of computer assisted sperm analysis (CASA) were performed. There were no significant differences between the total motility of the control (60.48%) and treatments (49.0; 47.8 and 51.68%, respectively). The other kinetic parameters also showed no statistical difference. Thus, there is no need to use egg yolk in the freezing of P. brevis sperm when using a glucose and DMSO extender.(AU)

Influência de polissacarídeos sulfatados sobre a motilidade e morfologia espermática pós descongelação de Prochilodus brevis / (Influence of sulfated polysaccharides on post-thawing sperm motility and morphology of Prochilodus brevis

This research aims to verify the influence of sulfated polysaccharides, extracted from the skin of tilapia, on the after thawing motility and morphology of P. brevis sperm. For this, 17 males were hormonally induced to reproduce, through the application of two doses of pituitary carp extract, 0.4 and 4.0mg kg-1. After the seminal collection, objective analyzes were performed and samples with motility greater than 80% were selected to form the pools. Then, the pools were frozen in solution supplemented, or not, with different concentrations of glycosaminoglycans (GAGs): 0 (control); 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5 or 5.0mg mL-¹ (total of 10 treatments). The samples were placed in 0.25 mL French straws and submitted to an equilibrium time of 10 minutes at 4 ºC. Then, they were kept in the dry shipper for 15 minutes and finally stored in liquid nitrogen. After 15 days, samples were thawed in a water bath at 30 ºC for 16 seconds and evaluated for sperm motility and morphology. Thus, it was observed that the increase in GAGs caused a decrease in sperm motility, however the control and the concentration of 0.5mg mL-¹ presented very similar data. On the other hand, no decrease in normal sperm was observed with an increase in the concentration of GAGs. Therefore, it is concluded that the lowest concentration of GAGs is the most adequate to supplement the sperm freezing medium.

Influência de polissacarídeos sulfatados sobre a motilidade e morfologia espermática pós descongelação de Prochilodus brevis / (Influence of sulfated polysaccharides on post-thawing sperm motility and morphology of Prochilodus brevis

This research aims to verify the influence of sulfated polysaccharides, extracted from the skin of tilapia, on the after thawing motility and morphology of P. brevis sperm. For this, 17 males were hormonally induced to reproduce, through the application of two doses of pituitary carp extract, 0.4 and 4.0mg kg-1. After the seminal collection, objective analyzes were performed and samples with motility greater than 80% were selected to form the pools. Then, the pools were frozen in solution supplemented, or not, with different concentrations of glycosaminoglycans (GAGs): 0 (control); 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5 or 5.0mg mL-¹ (total of 10 treatments). The samples were placed in 0.25 mL French straws and submitted to an equilibrium time of 10 minutes at 4 ºC. Then, they were kept in the dry shipper for 15 minutes and finally stored in liquid nitrogen. After 15 days, samples were thawed in a water bath at 30 ºC for 16 seconds and evaluated for sperm motility and morphology. Thus, it was observed that the increase in GAGs caused a decrease in sperm motility, however the control and the concentration of 0.5mg mL-¹ presented very similar data. On the other hand, no decrease in normal sperm was observed with an increase in the concentration of GAGs. Therefore, it is concluded that the lowest concentration of GAGs is the most adequate to supplement the sperm freezing medium.(AU)

Maturation, spawning and larviculture of the mullet Mugil liza under laboratory conditions / Maturação, desova e larvicultura da tainha Mugil liza em laboratório

The technical feasibility for the sustainable production of a species depends mainly on the control of reproduction in captivity and the availability of juveniles. Studies were performed with the mullet Mugil liza in the 1980s, however this species was never produced commercially. With the development of aquaculture, the increasing demand for animal protein and the commercial value of the mullet roe, it was necessary to rear this species in the laboratory to produce juveniles in pilot scale. The broodstock, 4 females and 10 males, were kept in an indoor 12 m³ tank. Hormonal inductions were performed with the hormones pituitary extract of carp (PEC) and luteinizing hormone-releasing hormone analog (LHRHa); spawnings occurred between 54-57 h after the hormonal induction. Embryonated eggs of mullets were pelagic and translucent, with an average diameter of 846.29 ± 14.34 µm. The larvae hatched approximately 48 h after spawning with 2.95 ± 0.12 mm. The survival in the larviculture was estimated at 18.75%. Therefore, this study presents the technical methods for the capture, transport, acclimatization, maturation, induced spawning and hatchery of the mullet M. liza in the laboratory.(AU)

A viabilidade técnica para a produção sustentável de uma espécie depende principalmente do controle da reprodução em cativeiro e da disponibilidade de juvenis. Estudos foram realizados com a tainha Mugil liza nos anos 80, entretanto, essa espécie não chegou a ser produzida comercialmente. Com o desenvolvimento da aquicultura, com a crescente demanda por proteína animal, além do valor comercial agregado nas gônadas dessa espécie, surgiu à necessidade de tentar produzi-la em laboratório, desta vez com o objetivo de produzir juvenis em escala-piloto. Os reprodutores, 4 fêmeas e 10 machos, foram mantidos em tanque de 12 m³. As induções hormonais foram realizadas com os hormônios extrato bruto de hipófise de carpa (EBHC) e um análogo do hormônio liberador do hormônio luteinizante (LHRHa); as desovas ocorreram 54-57 h depois da indução hormonal. Os ovos embrionados das tainhas são pelágicos e translúcidos, com um diâmetro médio de 846.29 ± 14.34 µm. As larvas eclodiram aproximadamente 48h após a desova com 2.95 ± 0.12 mm. A sobrevivência desde a estocagem dos ovos até a despesca dos juvenis foi estimada em 18,75%. Sendo assim, este estudo apresenta os métodos técnicos para a captura, transporte, aclimatação, maturação, desova induzida e larvicultura da tainha M. liza em laboratório.(AU)

Maturation, spawning and larviculture of the mullet Mugil liza under laboratory conditions / Maturação, desova e larvicultura da tainha Mugil liza em laboratório

The technical feasibility for the sustainable production of a species depends mainly on the control of reproduction in captivity and the availability of juveniles. Studies were performed with the mullet Mugil liza in the 1980s, however this species was never produced commercially. With the development of aquaculture, the increasing demand for animal protein and the commercial value of the mullet roe, it was necessary to rear this species in the laboratory to produce juveniles in pilot scale. The broodstock, 4 females and 10 males, were kept in an indoor 12 m³ tank. Hormonal inductions were performed with the hormones pituitary extract of carp (PEC) and luteinizing hormone-releasing hormone analog (LHRHa); spawnings occurred between 54-57 h after the hormonal induction. Embryonated eggs of mullets were pelagic and translucent, with an average diameter of 846.29 ± 14.34 µm. The larvae hatched approximately 48 h after spawning with 2.95 ± 0.12 mm. The survival in the larviculture was estimated at 18.75%. Therefore, this study presents the technical methods for the capture, transport, acclimatization, maturation, induced spawning and hatchery of the mullet M. liza in the laboratory.

A viabilidade técnica para a produção sustentável de uma espécie depende principalmente do controle da reprodução em cativeiro e da disponibilidade de juvenis. Estudos foram realizados com a tainha Mugil liza nos anos 80, entretanto, essa espécie não chegou a ser produzida comercialmente. Com o desenvolvimento da aquicultura, com a crescente demanda por proteína animal, além do valor comercial agregado nas gônadas dessa espécie, surgiu à necessidade de tentar produzi-la em laboratório, desta vez com o objetivo de produzir juvenis em escala-piloto. Os reprodutores, 4 fêmeas e 10 machos, foram mantidos em tanque de 12 m³. As induções hormonais foram realizadas com os hormônios extrato bruto de hipófise de carpa (EBHC) e um análogo do hormônio liberador do hormônio luteinizante (LHRHa); as desovas ocorreram 54-57 h depois da indução hormonal. Os ovos embrionados das tainhas são pelágicos e translúcidos, com um diâmetro médio de 846.29 ± 14.34 µm. As larvas eclodiram aproximadamente 48h após a desova com 2.95 ± 0.12 mm. A sobrevivência desde a estocagem dos ovos até a despesca dos juvenis foi estimada em 18,75%. Sendo assim, este estudo apresenta os métodos técnicos para a captura, transporte, aclimatação, maturação, desova induzida e larvicultura da tainha M. liza em laboratório.

Leporinus elongatus induced spawning using carp pituitary extract or mammalian GnRH analogue combined with dopamine receptor antagonists

Several studies have been developed to support the replacement of the crude carp pituitary extract (CPE) by synthetic products for induced reproduction of South American rheophilic species. However, results have been quite heterogeneous and there is no consensus or a routine use of synthetic products in these species. Thus, the aim of this study was to evaluate the ovulatory process in L. elongatus using different protocols of hormonal induction. Thus, fifteen wild mature females maintained at the Experimental Fish Station, Salto Grande, SP, Brazil were submitted to three different hormonal treatments: CPE (fractioned dose: 0.5 and 5.0 mg kg-1 ); mGnRHa (single dose: 3.5 µg kg-1 ) and mGnRHa (single dose: 5.0 µg kg-1 ). The spawning rate and absolute fecundity were similar among the treatments, but fertility rates were higher for CPE treatment (23.60 ± 9.40) then for mGnRHa treatments (close to or zero zero). Although females ovulated in all treatments, none of them provided viable embryos, showing hatching rates close to zero or zero. Both mGnRHa treatments were more potent for inducing the ovulatory process then CPE treatment, which was evidenced by the fact that the formers showed higher volume density of postovulatory follicles (POF). Accordingly, E2 and 17α-OHP plasma levels were higher for the mGnRHa treated females compared to the CPE one at the time of ovulation. In this study we confirmed previous scientific evidence that, regardless of whether promoting ovulation, the use of conventional CPE and GnRH doses are not appropriate for some South American migratory species, due to the nonattainment of viable embryos. Moreover, we have brought new information about the relationship between reproductive performance and gonadal steroids concentrations using different hormonal therapies, contributing to understand the reasons for Leporinus elongatus embryo loss in induced spawning.

Leporinus elongatus induced spawning using carp pituitary extract or mammalian GnRH analogue combined with dopamine receptor antagonists

Several studies have been developed to support the replacement of the crude carp pituitary extract (CPE) by synthetic products for induced reproduction of South American rheophilic species. However, results have been quite heterogeneous and there is no consensus or a routine use of synthetic products in these species. Thus, the aim of this study was to evaluate the ovulatory process in L. elongatus using different protocols of hormonal induction. Thus, fifteen wild mature females maintained at the Experimental Fish Station, Salto Grande, SP, Brazil were submitted to three different hormonal treatments: CPE (fractioned dose: 0.5 and 5.0 mg kg-1 ); mGnRHa (single dose: 3.5 µg kg-1 ) and mGnRHa (single dose: 5.0 µg kg-1 ). The spawning rate and absolute fecundity were similar among the treatments, but fertility rates were higher for CPE treatment (23.60 ± 9.40) then for mGnRHa treatments (close to or zero zero). Although females ovulated in all treatments, none of them provided viable embryos, showing hatching rates close to zero or zero. Both mGnRHa treatments were more potent for inducing the ovulatory process then CPE treatment, which was evidenced by the fact that the formers showed higher volume density of postovulatory follicles (POF). Accordingly, E2 and 17α-OHP plasma levels were higher for the mGnRHa treated females compared to the CPE one at the time of ovulation. In this study we confirmed previous scientific evidence that, regardless of whether promoting ovulation, the use of conventional CPE and GnRH doses are not appropriate for some South American migratory species, due to the nonattainment of viable embryos. Moreover, we have brought new information about the relationship between reproductive performance and gonadal steroids concentrations using different hormonal therapies, contributing to understand the reasons for Leporinus elongatus embryo loss in induced spawning.(AU)

Semen characterization of tambaqui (Colossoma macropomum) during the breeding season / Caracterização do sêmen de tambaqui (Colossoma macropomum) durante período reprodutivo

This study aims to qualitatively and quantitatively characterize the semen of tambaqui (Colossoma macropomum) during the breeding season. Three samplings were made at regular intervals of approximately 30 days in the 2012/2013 breeding season. Ten adult males of at least three years old age were selected. To collect the semen, hormonal induction was performed with carp pituitary extract, and extraction occurred by abdominal massage. After collection, the semen was subjected to evaluation of motility, vigor, lifetime, ejaculate volume, and sperm concentration. Analysis of variance (ANOVA) and Tukey test were applied on quantitative variables, while the Freedman test was used with 0.05 significance on qualitative variables. No significant differences (p>0.05) were observed for most parameters evaluated. Sperm concentration significantly varied between samples (14.5, 3.7, and 8.3x109 sperm/mL) and lifetime decreased significantly between the second and third samples (267.8 e 98.5 seconds, respectively).

Objetivou-se com o presente trabalho caracterizar qualitativamente e quantitativamente o sêmen de tambaqui durante o período reprodutivo. Foram realizadas três coletas, com intervalos regulares de aproximadamente 30 dias no período reprodutivo de 2012/2013. Foram utilizados dez reprodutores de no mínimo três anos de idade. Para a coleta de sêmen foi realizada indução hormonal com extrato de hipófise de carpa, e a extração do sêmen ocorreu por massagem abdominal. Após coleta, o sêmen foi submetido a avaliações de motilidade, vigor, tempo de vida, volume do ejaculado e concentração espermática. As variáveis quantitativas foram submetidas à análise de variância (ANOVA) e teste de Tukey, e as variáveis qualitativas ao teste de Friedman, com significância de 0,05. Não foram observadas diferenças significativas (p>0,05) para a maioria dos parâmetros avaliados. A concentração espermática variou significativamente entre as coletas (14,5, 3,7, e 8,3x109 espermatozoides/mL) e o tempo de vida diminuiu significativamente entre a segunda e terceira coletas (267,8 e 98,5 segundos, respectivamente).

Semen characterization of tambaqui (Colossoma macropomum) during the breeding season / Caracterização do sêmen de tambaqui (Colossoma macropomum) durante período reprodutivo

This study aims to qualitatively and quantitatively characterize the semen of tambaqui (Colossoma macropomum) during the breeding season. Three samplings were made at regular intervals of approximately 30 days in the 2012/2013 breeding season. Ten adult males of at least three years old age were selected. To collect the semen, hormonal induction was performed with carp pituitary extract, and extraction occurred by abdominal massage. After collection, the semen was subjected to evaluation of motility, vigor, lifetime, ejaculate volume, and sperm concentration. Analysis of variance (ANOVA) and Tukey test were applied on quantitative variables, while the Freedman test was used with 0.05 significance on qualitative variables. No significant differences (p>0.05) were observed for most parameters evaluated. Sperm concentration significantly varied between samples (14.5, 3.7, and 8.3x109 sperm/mL) and lifetime decreased significantly between the second and third samples (267.8 e 98.5 seconds, respectively).(AU)

Objetivou-se com o presente trabalho caracterizar qualitativamente e quantitativamente o sêmen de tambaqui durante o período reprodutivo. Foram realizadas três coletas, com intervalos regulares de aproximadamente 30 dias no período reprodutivo de 2012/2013. Foram utilizados dez reprodutores de no mínimo três anos de idade. Para a coleta de sêmen foi realizada indução hormonal com extrato de hipófise de carpa, e a extração do sêmen ocorreu por massagem abdominal. Após coleta, o sêmen foi submetido a avaliações de motilidade, vigor, tempo de vida, volume do ejaculado e concentração espermática. As variáveis quantitativas foram submetidas à análise de variância (ANOVA) e teste de Tukey, e as variáveis qualitativas ao teste de Friedman, com significância de 0,05. Não foram observadas diferenças significativas (p>0,05) para a maioria dos parâmetros avaliados. A concentração espermática variou significativamente entre as coletas (14,5, 3,7, e 8,3x109 espermatozoides/mL) e o tempo de vida diminuiu significativamente entre a segunda e terceira coletas (267,8 e 98,5 segundos, respectivamente).(AU)

Astyanax altiparanae ovarian maturation after spawning in water recycling systems / Maturação ovariana de Astyanax altiparanae após desova em sistema de recirculação de água

In this study, we evaluated the possibility of obtaining successive Astyanax altiparanae spawns under laboratory conditions. In order to do so, 104 specimens were randomly distributed into four boxes (10 females and 16 males each) and kept in a recirculation system at an average temperature of 29.24 ± 0.42 °C, under natural photoperiod, for 30 days. On the onset of the experiment, males and females were induced for reproduction with a 6 mg kg-1 carp pituitary extract dose. After that, ovary (for gonadosomatic index and stereological assessment) and blood samples (for steroid evaluation) were collected from eight females (two per box) at the following moments: immediately after hormonal induction (day 0) and on days 1, 6, 16 and 30 after spawning. On day 6, spawned females presented complete mature ovaries similar to those on day 0 and, in this period, we did not observe postovulatory complexes, indicating that their resorption happened very fast. Concomitantly, the steroid levels increased gradually up to day 6, which corroborated an intense vitellogenic activity in this period. This study has demonstrated that A. altiparanae females are suitable for induced spawning within six days after spawning, when kept at 29.24 ± 0.42 °C, in a system maintained with recirculated water.

Neste estudo, avaliamos a possibilidade de obtenção de desovas sucessivas de Astyanax altiparanae mantidos em biotério. Para isso, 104 indivíduos foram distribuídos aleatoriamente em quatro caixas (10 fêmeas e 16 machos em cada) e mantidos a uma temperatura média de 29,24 ± 0,42 °C, sob fotoperíodo natural, por 30 dias. No início do experimento, machos e fêmeas foram induzidos à reprodução com uma dose de extrato de hipófise de carpa (6 mg kg-1). Posteriormente, amostras de ovário (para cálculo do índice gonadosomático e avaliação estereológica) e de sangue (para avaliação de esteroides) foram coletadas de oito fêmeas (duas por caixa) nos seguintes períodos: imediatamente após a indução hormonal (dia 0) e 1, 6, 16 e 30 dias após a desova. No dia 6, as fêmeas desovadas já apresentavam ovários maduros completos similares aos descritos no dia 0. No dia 6 não foram mais observados complexos pós-ovulatórios, indicando que a sua reabsorção foi relativamente rápida. Concomitantemente, os níveis de esteroides aumentaram gradativamente até o dia 6, corroborando uma atividade vitelogênica intensa neste período. Neste estudo demonstramos que fêmeas de A. altiparanae apresentam-se aptas para indução hormonal seis dias após a reprodução, quando mantida a 29,24 ± 0,42 °C, em um sistema mantido com água recirculada.

Astyanax altiparanae ovarian maturation after spawning in water recycling systems / Maturação ovariana de Astyanax altiparanae após desova em sistema de recirculação de água

In this study, we evaluated the possibility of obtaining successive Astyanax altiparanae spawns under laboratory conditions. In order to do so, 104 specimens were randomly distributed into four boxes (10 females and 16 males each) and kept in a recirculation system at an average temperature of 29.24 ± 0.42 °C, under natural photoperiod, for 30 days. On the onset of the experiment, males and females were induced for reproduction with a 6 mg kg-1 carp pituitary extract dose. After that, ovary (for gonadosomatic index and stereological assessment) and blood samples (for steroid evaluation) were collected from eight females (two per box) at the following moments: immediately after hormonal induction (day 0) and on days 1, 6, 16 and 30 after spawning. On day 6, spawned females presented complete mature ovaries similar to those on day 0 and, in this period, we did not observe postovulatory complexes, indicating that their resorption happened very fast. Concomitantly, the steroid levels increased gradually up to day 6, which corroborated an intense vitellogenic activity in this period. This study has demonstrated that A. altiparanae females are suitable for induced spawning within six days after spawning, when kept at 29.24 ± 0.42 °C, in a system maintained with recirculated water.(AU)

Neste estudo, avaliamos a possibilidade de obtenção de desovas sucessivas de Astyanax altiparanae mantidos em biotério. Para isso, 104 indivíduos foram distribuídos aleatoriamente em quatro caixas (10 fêmeas e 16 machos em cada) e mantidos a uma temperatura média de 29,24 ± 0,42 °C, sob fotoperíodo natural, por 30 dias. No início do experimento, machos e fêmeas foram induzidos à reprodução com uma dose de extrato de hipófise de carpa (6 mg kg-1). Posteriormente, amostras de ovário (para cálculo do índice gonadosomático e avaliação estereológica) e de sangue (para avaliação de esteroides) foram coletadas de oito fêmeas (duas por caixa) nos seguintes períodos: imediatamente após a indução hormonal (dia 0) e 1, 6, 16 e 30 dias após a desova. No dia 6, as fêmeas desovadas já apresentavam ovários maduros completos similares aos descritos no dia 0. No dia 6 não foram mais observados complexos pós-ovulatórios, indicando que a sua reabsorção foi relativamente rápida. Concomitantemente, os níveis de esteroides aumentaram gradativamente até o dia 6, corroborando uma atividade vitelogênica intensa neste período. Neste estudo demonstramos que fêmeas de A. altiparanae apresentam-se aptas para indução hormonal seis dias após a reprodução, quando mantida a 29,24 ± 0,42 °C, em um sistema mantido com água recirculada.(AU)

REPRODUÇÃO DE LAMBARI-DO-RABO-AMARELO (Astyanax lacustris) UTILIZANDO DIFERENTES INDUTORES

O lambari é um peixe com pequeno porte, que apresentou um aumento em sua produção nacional de 37% de 2015 a 2019 com um total de 661,019 Kg produzidos em 2019. O objetivo desse estudo foi avaliar a desova e características reprodutivas de diferentes indutores hormonais em adultos de lambari-do-rabo-amarelo (Astyanax lacustris). Foram utilizados 135 fêmeas (12,54 g ± 2,33 e 7,66 cm ± 0,63 cm) e 135 machos (5,83 g ± 0,39 g e 6,14 cm ± 0,64 cm), todos em idade reprodutiva. Foram avaliados três diferentes indutores: (i) 0,4 pellets de Ovopel®/kg de peso vivo; (ii) 0,5 mL de acetato de buserelina/kg de peso vivo; (iii) Extrato de Hipófise de Carpa (EHC) (5,5 mg de EHC/kg de peso vivo para fêmeas e 2,5 mg de EHC/kg de peso vivo para machos). A variável horas-grau foi maior (P<0,05) para o tratamento com Ovopel© e Acetato de buserelina em relação ao tratamento com EHC. O número de larvas por peso das fêmeas foi maior com a utilização do Ovopel®. Fecundidade absoluta e fecundidade relativa foram maiores para os tratamentos com Ovopel® e EHC. A maior taxa de fertilização ocorreu no tratamento com acetato de buserelina. A taxa de eclosão foi maior para o acetato de buserelina e Ovopel®. A utilização de Ovopel® proporcionou melhores resultados para a reprodução de A. lacustris quando comparados ao EHC e acetato de buserelina.

Lambari is a small fish, which had an increase of 37% in its production from 2015 to 2019 with a total amount of 661.019 Kg produced in the year of 2019. The objective of this study was to evaluate the spawning and reproductive characteristics of different spawning inducers in adults of yellow-tailed lambari (Astyanax lacustris). A total of 135 females (12.54 g ± 2.33 and 7.66 cm ± 0.63 cm) and 135 males (5.83 g ± 0.39 g and 6.14 cm ± 0.64 cm), all of reproductive age, were used. Three different inducers were evaluated: (i) 0.4 pellets of Ovopel®/kg of body weight; (ii) 0.5 ml of buserelin acetate/kg of body weight; (iii) Carp's Pituitary Extract (CPE) (5.5 mg CPE/kg body weight for females and 2.5 mg of CPE/kg of body weight for males). The variable hour-degree was higher (P <0.05) for the treatment with Ovopel © and buserelin acetate compared to the treatment with CPE. There number of larvae per female body weight was greater when using Ovopel®. Absolute fecundity (AF) and relative fecundity (RF) were higher for treatments with Ovopel® and CPE. The higher fertilization rate occurred in the treatment with buserelin acetate. The hatching rate was higher for buserelin acetate and Ovopel®. The use of Ovopel® provided greater results in the reproduction of A. lacustris when compared to CPE and buserelin acetate.

QUALIDADE DO SÊMEN DE TAMBAQUI (Colossoma macropomum) CRIOPRESERVADO EM DIFERENTES CONCENTRAÇÕES DE GEMA DE OVO

Abstract The aim of this study was to evaluate the effect of egg yolk (EY) addition on the sperm kinects after cryopreservation. We used twenty tambaqui males (n = 4 pools), which were induced homonally to spermiation with carp pituitary extract. Fourteen hours after induction, a seminal collection was performed. The semen from each pool was diluted with Ringer added 10% DMSO on the presence or absence of egg yolk (T1: no additional EY; T2: 5% EY; and T3: 10% EY). The treated semen was loaded in 0.5 mL straws, frozen in a nitrogen vapor vessel (dry shipper) (30 min / -153 °C), and then transferred to liquid nitrogen. Straws were thawed in a water bath at 37 °C / 30 seconds. The analyses of motility rate (%) and sperm curvilinear velocity (µm/s) were performed on computerized system (CASA). Data were expressed as mean ± standard deviation, and Tukey test was applied (P 0.05). There was a significant reduction in the percentage of motile spermatozoa and curvilinear velocity after the addition of egg yolk to the crioprotection solution, regardless of the concentration. Therefore, the addition of EY to Ringer + DMSO had negative effect on motility of tambaqui frozen sperm.

Resumo O objetivo deste trabalho foi avaliar o efeito da adição de gema de ovo (GO) sobre a cinética dos espermatozoides de tambaquis após a criopreservação. Utilizaram-se vinte machos de tambaquis (n= 4 pools), que foram induzidos hormonalmente com extrato hipofisário de carpa, para espermiação. Quatorze horas após a indução, realizou-se a coleta seminal. O sêmen de cada pool foi diluído em Ringer adicionado de 10% de dimetilsulfóxido (DMSO) acrescido ou não de GO (T1: sem acréscimo de GO; T2: com 5% de GO e T3: com 10% de GO). O sêmen tratado foi envasado em palhetas de 0,5 mL, congelado em vapor de nitrogênio líquido (dry shipper-30 min/-153 °C) e posteriormente transferidas para nitrogênio líquido. As palhetas foram descongeladas em banho-maria a 37 °C/30 segundos. A taxa de motilidade (%) e a velocidade curvilinear espermática (µm/s) foram analisadas em sistema computadorizado (CASA). Os dados foram expressos em média ± desvio padrão e foi aplicado o teste de Tukey (P 0,05). Houve uma redução significativa na porcentagem de espermatozoides móveis e velocidade curvilinear após a adição de GO independente da concentração. Logo, a adição de GO ao Ringer + DMSO teve efeito negativo sobre a motilidade do sêmen congelado de tambaqui.

COLETAS SUCESSIVAS DE SÊMEN DE Leiarius marmoratus: CARACTERÍSTICAS QUANTITATIVAS E QUALITATIVAS

O objetivo do estudo foi avaliar as características reprodutivas de machos de Leiarius marmoratus em coletas sucessivas no mesmo período reprodutivo. Foram utilizados 10 machos adultos com peso médio de 3,24±1,24 kg, os quais foram avaliados em quatro coletas sucessivas com intervalo de 10 dias. Para estas coletas, sete machos foram induzidos com extrato de hipófise de carpa na posologia de 3,0 mg/kg de peso corporal em duas aplicações (30 e 70%) com intervalo de 10 horas; e em três peixes foi aplicado apenas soro fisiológico (controle). Após 180 horas-grau da segunda aplicação, foram avaliados parâmetros espermáticos computadorizados, concentração espermática, taxa de sobrevivência e taxa de normalidade. Apenas um macho não induzido liberou um pequeno volume de sêmen (0,33 mL) e apenas na primeira coleta. Dos sete machos induzidos, cinco machos liberaram sêmen nas quatro coletas, porém apenas quatro foram considerados nas análises estatísticas (n=4). Não houve diferença no volume de sêmen da primeira coleta (0,63 mL) em relação as demais coletas (0,59 a 1,38 mL). Maior (P<0,05) concentração foi observado na primeira coleta (1,98±0,34 x109 espermatozoides mL-1) em relação as demais coletas (0,35±0,14 x109 a 0,92±0,09 x109 espermatozoides mL-1). A motilidade espermática, velocidade curvilinear, retilinearidade e normalidade não diferiram entre as coletas. Os valores de velocidade média de deslocamento, oscilação, linearidade, progressão e sobrevivência mantiveram-se iguais (P>0,05) até a segunda coleta. Os valores de velocidade em linha reta e frequência de batimento transposto foram maiores (P<0,05) na primeira coleta em relação as demais. Conclui-se que há uma redução quantitativa do sêmen das duas primeira para as demais coletas, mas em geral sem grandes alterações nas características qualitativas nas quatro coletas.

The objective of the study was to evaluate the reproductive characteristics of Leiarius marmoratus males in successive collections in the same reproductive period. Ten adult males with an average weight of 3.24±1.24 kg were used, which were adopted in four successive collections with an interval of 10 days. For these collections, seven males were induced with carp pituitary extract at a dose of 3.0 mg / kg of body weight in two applications (30 and 70%) with an interval of 10 hours; and in three fish only saline (control) was projected. After 180 thermal units accumulated from the second application, computerized sperm parameters, sperm concentration, survival rate and normality rate were evaluated. Only one uninduced male released a small volume of semen (0.33 mL) and only in the first collection. Of the seven males induced, five males released semen in the four collections, although only four were considered in the statistical analyzes (n = 4). There was no difference in the semen volume of the first collection (0.63 mL) in relation to the other collections (0.59 to 1.38 mL). Higher (P<0.05) concentration was observed in the first collection (1.98±0.34 x109 sperm mL-1) in relation to the other collections (0.35±0.14 x109 at 0.92±0.09 x109 sperm mL-1). Sperm motility, curvilinear velocity, straightness and normality did not differ between collections. The mean average path velocity, oscillation, linearity, progression and survival values remained the same (P> 0.05) until the second collection. The values of straight line velocity and frequency of transposed beating were higher (P <0.05) in the first collection in relation to the others. It is concluded that there is a quantitative reduction of semen from the first two to the other collections, but in general without major changes in the qualitative characteristics in the four collections

Qualidade do sêmen de tambaqui(Colossoma macropomum) criopreservado em diferentes concentrações de gema de ovo / Quality of tambaqui (Colossoma macropomum) semen cryopreserved in different concentrations of egg yolk

O objetivo deste trabalho foi avaliar o efeito da adição de gema de ovo (GO) sobre a cinética dos espermatozoides de tambaquis após a criopreservação. Utilizaram-se vinte machos de tambaquis (n= 4 pools), que foram induzidos hormonalmente com extrato hipofisário de carpa, para espermiação. Quatorze horas após a indução, realizou-se a coleta seminal. O sêmen de cada pool foi diluído em Ringer adicionado de 10% de dimetilsulfóxido (DMSO) acrescido ou não de GO (T1: sem acréscimo de GO; T2: com 5% de GO e T3: com 10% de GO). O sêmen tratado foi envasado em palhetas de 0,5 mL, congelado em vapor de nitrogênio líquido (dry shipper-30 min/-153 °C) e posteriormente transferidas para nitrogênio líquido. As palhetas foram descongeladas em banho-maria a 37 °C/30 segundos. A taxa de motilidade (%) e a velocidade curvilinear espermática (µm/s) foram analisadas em sistema computadorizado (CASA). Os dados foram expressos em média ± desvio padrão e foi aplicado o teste de Tukey (P<0,05). Houve uma redução significativa na porcentagem de espermatozoides móveis e velocidade curvilinear após a adição de GO independente da concentração. Logo, a adição de GO ao Ringer + DMSO teve efeito negativo sobre a motilidade do sêmen congelado de tambaqui

The aim of this study was to evaluate the effect of egg yolk (EY) addition on the sperm kinects after cryopreservation. We used twenty tambaqui males (n = 4 pools), which were induced homonally to spermiation with carp pituitary extract. Fourteen hours after induction, a seminal collection was performed. The semen from each pool was diluted with Ringer added 10% DMSO on the presence or absence of egg yolk (T1: no additional EY; T2: 5% EY; and T3: 10% EY). The treated semen was loaded in 0.5 mL straws, frozen in a nitrogen vapor vessel (dry shipper) (30 min / -153 °C), and then transferred to liquid nitrogen. Straws were thawed in a water bath at 37 °C / 30 seconds. The analyses of motility rate (%) and sperm curvilinear velocity (µm/s) were performed on computerized system (CASA). Data were expressed as mean ± standard deviation, and Tukey test was applied (P<0.05). There was a significant reduction in the percentage of motile spermatozoa and curvilinear velocity after the addition of egg yolk to the crioprotection solution, regardless of the concentration. Therefore, the addition of EY to Ringer + DMSO had negative effect on motility of tambaqui frozen sperm