Determination of the ideal volume of activating solution and the optimal ratio of spermatozoa per oocyte for Prochilodus brevis fertilization.

Prochilodus brevis is a rheophilic species with a threatened natural population that promotes studies aimed at optimizing reproduction in captivity. The correct quantity of inseminating dose and activating solution volume significantly improves fertilization rates, thereby increasing productivity in captivity. The objective of this study was to determine the proportion of sperm per oocyte and the ideal volume of activating solution to be used in the assisted fertilization of P. brevis. Gametes were collected and fertilization performed in two steps. In step 1, the ideal proportion of spermatozoa was determined based on the fertilization rate:oocyte by testing six doses of semen: D1 = 30 × 103, D2 = 150 × 103, D3 = 300 × 103, D4 = 3 × 106, D5 = 5 × 106, and D6 = 10 × 106. In step 2, the fertilization and hatching rates were evaluated using different volumes of activating solution (V1 - 25 ml, V2 - 50 ml, V3 - 75 ml,V4 - 100 ml, V5 - 125 ml, and V6 - 150 ml). A linear regression equation was estimated from steps 1 and 2. The Student-Newman-Keuls test was used to compare the means. In step 1, the percentage of fertilization increased linearly, reaching a plateau of 51.69%. In step 2, the best fertilization rates were obtained with an estimated ideal volume of 75.64 ml per 2 ml of oocytes. Therefore, the proportion of 928,410.29 sperm:oocyte, associated with the volume of 75.64 ml of water per 2 ml of oocytes, provided the maximum reproductive performance for P. brevis.

Capacidade fecundante do sêmen congelado de Prochilodus brevis / Fertilizing capacity of the cryopreserved sperm of Prochilodus brevis

Background: Seminal cryopreservation is a technique that optimizes aquacultural production, as it requires less breedingand enables reproduction outside of the breeding season. This technique also helps to preserve species, thus reducing thepressure on the natural stocks. Several studies have sought to develop freezing protocols that result in semen of a goodquality. However, some studies do not evaluate the ability of frozen semen to produce viable larvae. Therefore, the aimof this study was to verify the fertilizing capacity of the frozen semen of Prochilodus brevis.Materials, Methods & Results: Semen from twenty adult males of the Brazilian bocachico was collected and evaluatedto establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, membrane integrity, pH,and concentration. Six pools were formed, each of which was diluted in a freezing medium containing 5% glucose with10% dimethyl sulfoxide (DMSO) or 5% glucose with 10% methyl glycol (MG). The samples were loaded into 0.25 mLFrench straws, frozen in a dry shipper, and stored in a liquid nitrogen canister. The semen was then thawed and evaluatedto establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, and membrane integrity.For the fertilization test, four females were used. The oocytes from each female were divided into three batches and fertilized with either fresh or cryopreserved semen. The rates of fertilization, hatching, and larval survival were then measured.Data were expressed as the mean ± standard deviation and analyzed using SAS (2002). The frozen semen with glucose +DMSO was significantly higher (P < 0.001) than the frozen semen with glucose + MG, in all seminal quality parametersevaluated (63.95 ± 15.88% and 25.36 ± 3.53% for the motility, 36.38 ± 7.02 μm.s-1 and 20.45 ± 2.84 μm.s-1 for the curvilinear velocity, 19.26 ± 2.74 μm.s-1 and...(AU)

Utilização de soluções de natureza orgânicas em diferentes concentrações, na ativação do sêmen de curimatã comum / Use of organic natural solutions at different concentrations, on activation of the common curimatem semen

A determinação da solução adequada à ativação da motilidade espermática torna-se uma questão importante em trabalhos envolvendo o sêmen de peixes. Assim, o objetivo do trabalho foi avaliar a atuação de soluções ativadoras, de composição orgânica, em diferentes concentrações sobre a cinética do sêmen in natura de Prochilodus brevis. O sêmen foi coletado e foram feitas análises de motilidade espermática, velocidade curvilinear (VCL), velocidade em linha reta (VSL) e velocidade média do percurso (VAP) e duração da motilidade espermática. A glicose 200 mM tendeu a apresentar maiores valores que os demais (p<0,05), apresentando diferença em relação à mesma concentração com frutose na VSL. Conclui-se que a glicose 200 mM é o mais recomendado para a ativação espermática de P. brevis.(AU)

Determination of the appropriate solution to the activation of sperm motility becomes an important issue in studies involving fish semen. Thus, the objective of this work was to evaluate the performance of activating solutions, of organic composition, in different concentrations on the kinetics of the semen in natura of Prochilodus brevis. Semen was collected and analyzes of sperm motility, curvilinear velocity (VCL), velocity in the straight line (VSL) and mean velocity of the trajectory (VAP) and duration of sperm motility were made. The 200 mM glucose tended to present higher values than the others (p<0.05), presenting difference in relation to the same concentration with fructose in VSL. It is concluded that 200 mM glucose is the most recommended for the sperm activation of P. brevis.(AU)

Capacidade fecundante do sêmen congelado de Prochilodus brevis / Fertilizing capacity of the cryopreserved sperm of Prochilodus brevis

Background: Seminal cryopreservation is a technique that optimizes aquacultural production, as it requires less breedingand enables reproduction outside of the breeding season. This technique also helps to preserve species, thus reducing thepressure on the natural stocks. Several studies have sought to develop freezing protocols that result in semen of a goodquality. However, some studies do not evaluate the ability of frozen semen to produce viable larvae. Therefore, the aimof this study was to verify the fertilizing capacity of the frozen semen of Prochilodus brevis.Materials, Methods & Results: Semen from twenty adult males of the Brazilian bocachico was collected and evaluatedto establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, membrane integrity, pH,and concentration. Six pools were formed, each of which was diluted in a freezing medium containing 5% glucose with10% dimethyl sulfoxide (DMSO) or 5% glucose with 10% methyl glycol (MG). The samples were loaded into 0.25 mLFrench straws, frozen in a dry shipper, and stored in a liquid nitrogen canister. The semen was then thawed and evaluatedto establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, and membrane integrity.For the fertilization test, four females were used. The oocytes from each female were divided into three batches and fertilized with either fresh or cryopreserved semen. The rates of fertilization, hatching, and larval survival were then measured.Data were expressed as the mean ± standard deviation and analyzed using SAS (2002). The frozen semen with glucose +DMSO was significantly higher (P < 0.001) than the frozen semen with glucose + MG, in all seminal quality parametersevaluated (63.95 ± 15.88% and 25.36 ± 3.53% for the motility, 36.38 ± 7.02 μm.s-1 and 20.45 ± 2.84 μm.s-1 for the curvilinear velocity, 19.26 ± 2.74 μm.s-1 and...

Utilização de soluções de natureza orgânicas em diferentes concentrações, na ativação do sêmen de curimatã comum / Use of organic natural solutions at different concentrations, on activation of the common curimatem semen

A determinação da solução adequada à ativação da motilidade espermática torna-se uma questão importante em trabalhos envolvendo o sêmen de peixes. Assim, o objetivo do trabalho foi avaliar a atuação de soluções ativadoras, de composição orgânica, em diferentes concentrações sobre a cinética do sêmen in natura de Prochilodus brevis. O sêmen foi coletado e foram feitas análises de motilidade espermática, velocidade curvilinear (VCL), velocidade em linha reta (VSL) e velocidade média do percurso (VAP) e duração da motilidade espermática. A glicose 200 mM tendeu a apresentar maiores valores que os demais (p<0,05), apresentando diferença em relação à mesma concentração com frutose na VSL. Conclui-se que a glicose 200 mM é o mais recomendado para a ativação espermática de P. brevis.

Determination of the appropriate solution to the activation of sperm motility becomes an important issue in studies involving fish semen. Thus, the objective of this work was to evaluate the performance of activating solutions, of organic composition, in different concentrations on the kinetics of the semen in natura of Prochilodus brevis. Semen was collected and analyzes of sperm motility, curvilinear velocity (VCL), velocity in the straight line (VSL) and mean velocity of the trajectory (VAP) and duration of sperm motility were made. The 200 mM glucose tended to present higher values than the others (p<0.05), presenting difference in relation to the same concentration with fructose in VSL. It is concluded that 200 mM glucose is the most recommended for the sperm activation of P. brevis.

Efeito de vitaminas e aminoácidos como suplementação da solução crioprotetora para a criopreservação do sêmen de tambaqui (Colossoma macropomum) / Effect of vitamins and amino acids as cryoprotectant solution supplementation for the cryopreservation of tambaqui semen (Colossoma macropomum)

Background: The addition of antioxidant substances to a cryoprotective solution can increase its protective capacity, shielding spermatozoa from the oxidative stress caused by the seminal cryopreservation process. However, there is no record of a seminal cryopreservation protocol of tambaqui (Colossoma macropomum) using antioxidants as a supplement to the cryoprotective solution. Thus, the objective of this study was to evaluate the effects of adding vitamin C, vitamin E, cysteine, and/or taurine to the seminal cryopreservation of tambaqui.Materials, Methods & Results: Pools of semen (n = 10) were diluted in cryoprotective solutions supplemented with: vitamin C (T1), vitamin E (T2), vitamin C + vitamin E (T3), cysteine (T4), taurine (T5), and taurine + cysteine (T6). The control treatment (T7) was not supplemented. Diluted semen was loaded in 0.5 mL straws, frozen in a dry-shipper, stored in a cryogenic cylinder, and then thawed in a water bath (45ºC for eight seconds). The quality of fresh and cryopreserved semen was evaluated by measuring total motility, progressive motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity, and straightness using a computerized system of sperm analysis. Sperm membrane integrity parameters were analyzed using eosin-nigrosin staining, sperm morphology was assessed using pink bengal staining, and motility duration was measured by a digital timer. Data were analyzed using the statistical program SAS (2002) and the results were expressed as means ± standard error of the mean. The results showed that, in general, there was no significant increase in seminal quality when antioxidants were added to the cryoprotective solution. The T5 treatment promoted an increase (P < 0.05) in progressive motility when compared to T1 (6.33 ± 1.14% and 2.98 ± 0.88%, respectively). However, it did not differ significantly (P > 0.05) from the other treatments.[...](AU)

Efeito de vitaminas e aminoácidos como suplementação da solução crioprotetora para a criopreservação do sêmen de tambaqui (Colossoma macropomum) / Effect of vitamins and amino acids as cryoprotectant solution supplementation for the cryopreservation of tambaqui semen (Colossoma macropomum)

Background: The addition of antioxidant substances to a cryoprotective solution can increase its protective capacity, shielding spermatozoa from the oxidative stress caused by the seminal cryopreservation process. However, there is no record of a seminal cryopreservation protocol of tambaqui (Colossoma macropomum) using antioxidants as a supplement to the cryoprotective solution. Thus, the objective of this study was to evaluate the effects of adding vitamin C, vitamin E, cysteine, and/or taurine to the seminal cryopreservation of tambaqui.Materials, Methods & Results: Pools of semen (n = 10) were diluted in cryoprotective solutions supplemented with: vitamin C (T1), vitamin E (T2), vitamin C + vitamin E (T3), cysteine (T4), taurine (T5), and taurine + cysteine (T6). The control treatment (T7) was not supplemented. Diluted semen was loaded in 0.5 mL straws, frozen in a dry-shipper, stored in a cryogenic cylinder, and then thawed in a water bath (45ºC for eight seconds). The quality of fresh and cryopreserved semen was evaluated by measuring total motility, progressive motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity, and straightness using a computerized system of sperm analysis. Sperm membrane integrity parameters were analyzed using eosin-nigrosin staining, sperm morphology was assessed using pink bengal staining, and motility duration was measured by a digital timer. Data were analyzed using the statistical program SAS (2002) and the results were expressed as means ± standard error of the mean. The results showed that, in general, there was no significant increase in seminal quality when antioxidants were added to the cryoprotective solution. The T5 treatment promoted an increase (P 0.05) from the other treatments.[...]

Influência do tempo de resfriamento e soluções diluentes sobre a congelabilidade do sêmen de Prochilodus brevis / Influence of cooling time and diluents on the freezability of Prochilodus brevis semen

Background: Prochilodus brevis is a rheophilic fish of economic and ecological importance. However, anthropic action has made its population vulnerable. Thus, the development of reproductive biotechnologies, such as seminal conservation, is necessary to subsidize their fish farming. However, seminal collections are often performed in places with few laboratory resources, demanding studies to determine the maximum time for which sperm can be cooled, as well as its process until frozen. Thus, the present study aimed to evaluate the influence of cooling time and the presence of dilution solutions on cryopreservation of P. brevis semen.Material, Methods & Results: After seminal collection, nine pools were formed and analyzed for seminal pH, concentration, membrane integrity, morphology and spermatic kinetics - motility, curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). After the analysis of the pools in natura (control 1), they were processed as follows: 1)- immediate freezing (control 2); 2)- cooling: undiluted, diluted in coconut water powder (ACP-104) or diluted in 5% glucose, followed by cooling at different times (6, 12, 24 or 48 h); 3)- Post-refrigeration freezing: the pools were diluted in their respective diluents and 10% dimethyl sulfoxide. After 15 days, the samples were thawed and analyzed for the aforementioned parameters. For the cooled and post-thawed semen, a completely randomized design with 2 (diluent × cooling time) and 3 (storage form × cooling time and storage form × diluent) factors, respectively, was utilized. ANOVA and Dunnett tests were applied to compare the means. In case of seminal cooling, there was no difference (P > 0.05) in sperm motility between control 1 and the undiluted and diluted treatments in ACP-104 for up to 24 h. After 48 h, only the VCL of the sample diluted in ACP-104 was similar (P > 0.05) to that of control 1.[...](AU)

Influência do tempo de resfriamento e soluções diluentes sobre a congelabilidade do sêmen de Prochilodus brevis / Influence of cooling time and diluents on the freezability of Prochilodus brevis semen

Background: Prochilodus brevis is a rheophilic fish of economic and ecological importance. However, anthropic action has made its population vulnerable. Thus, the development of reproductive biotechnologies, such as seminal conservation, is necessary to subsidize their fish farming. However, seminal collections are often performed in places with few laboratory resources, demanding studies to determine the maximum time for which sperm can be cooled, as well as its process until frozen. Thus, the present study aimed to evaluate the influence of cooling time and the presence of dilution solutions on cryopreservation of P. brevis semen.Material, Methods & Results: After seminal collection, nine pools were formed and analyzed for seminal pH, concentration, membrane integrity, morphology and spermatic kinetics - motility, curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). After the analysis of the pools in natura (control 1), they were processed as follows: 1)- immediate freezing (control 2); 2)- cooling: undiluted, diluted in coconut water powder (ACP-104) or diluted in 5% glucose, followed by cooling at different times (6, 12, 24 or 48 h); 3)- Post-refrigeration freezing: the pools were diluted in their respective diluents and 10% dimethyl sulfoxide. After 15 days, the samples were thawed and analyzed for the aforementioned parameters. For the cooled and post-thawed semen, a completely randomized design with 2 (diluent × cooling time) and 3 (storage form × cooling time and storage form × diluent) factors, respectively, was utilized. ANOVA and Dunnett tests were applied to compare the means. In case of seminal cooling, there was no difference (P > 0.05) in sperm motility between control 1 and the undiluted and diluted treatments in ACP-104 for up to 24 h. After 48 h, only the VCL of the sample diluted in ACP-104 was similar (P > 0.05) to that of control 1.[...]

Cryopreservation of Prochilodus brevis semen: freezing media and thawing rates / Criopreservação do sêmen de Prochilodus brevis: meios de congelação e taxas de descongelação

Prochilodus brevis is a migratory fish, an important component of its river ecosystem and an appreciated animal in northeastern cuisine. However, human activities have threatened its survival. Thus, researchers have become interested in developing genetic material storage protocols, such as seminal cryopreservation. Therefore, determination of the appropriate freezing media and thawing rate is a fundamental step toward the use of this biotechnology in the production of common curimatã and for reducing risks to the species survival. As such, the aim of the current study was to evaluate the effect of different freezing media and thawing rates on the quality of cryopreserved semen from P. brevis. For this study, males received a single dose of pituitary extract of carp 18 hours before semen collection. The semen samples were diluted in 5% glucose + 10% methyl glycol (MG), 5% glucose + 10% dimethyl sulfoxide (DMSO), 0.9% NaCl + 10% methyl glycol, and 0.9% NaCl + 10% dimethyl sulfoxide, loaded into 0.25-mL straws and frozen in liquid nitrogen vapor. The semen from each treatment was thawed at three different thawing rates: 25 C for 30 sec, 30 C for 16 sec and 40 C for 12 sec. Motility, vitality and morphology analyses were performed by computer-assisted sperm analysis (CASA). The characteristics of the fresh sperm mostly resembled those found in the literature. For the parameter...(AU)

O Prochilodus brevis é um peixe reofílico, importante componente do ecossistema fluvial e apreciado na culinária nordestina. No entanto, ações antrópicas têm ameaçado sua sobrevivência. Desta forma, surge, nos pesquisadores, o interesse no desenvolvimento de protocolos de conservação do material genético, como a criopreservação seminal. Logo, a determinação do meio de congelação e da taxa de descongelação adequados, são passos fundamentais que possibilitarão a utilização dessa biotecnologia na produção de curimatã comum, reduzindo os riscos à sua sobrevivência. Portanto, o objetivo desse trabalho foi avaliar o efeito de diferentes meios de congelação e taxas de descongelação sobre a qualidade do sêmen criopreservado de P. brevis. Para isso, 18 horas antes da coleta de sêmen, os machos receberam dose única de extrato hipofisário de carpa. Cada animal foi sedado com solução à base de eugenol e o sêmen foi coletado. As amostras foram diluídas em quatro meios de congelação (5% Glicose + Metilglicol 10%; 5% Glicose + DMSO 10%; 0,9% NaCl + Metilglicol 10%; 0,9% NaCl + DMSO 10%) envasadas em palhetas de 0,25 mL e congeladas em vapor de nitrogênio líquido. O sêmen foi descongelado após sete dias em três taxas de descongelação: 25 C 30 s-1; 30 C 16 s-1; 40 C 12 s-1. Foram feitas as análises de motilidade, vitalidade e morfologia com auxílio de sistema automatizado de análise seminal...(AU)

Cryopreservation of Prochilodus brevis semen: freezing media and thawing rates / Criopreservação do sêmen de Prochilodus brevis: meios de congelação e taxas de descongelação

Prochilodus brevis is a migratory fish, an important component of its river ecosystem and an appreciated animal in northeastern cuisine. However, human activities have threatened its survival. Thus, researchers have become interested in developing genetic material storage protocols, such as seminal cryopreservation. Therefore, determination of the appropriate freezing media and thawing rate is a fundamental step toward the use of this biotechnology in the production of common curimatã and for reducing risks to the species survival. As such, the aim of the current study was to evaluate the effect of different freezing media and thawing rates on the quality of cryopreserved semen from P. brevis. For this study, males received a single dose of pituitary extract of carp 18 hours before semen collection. The semen samples were diluted in 5% glucose + 10% methyl glycol (MG), 5% glucose + 10% dimethyl sulfoxide (DMSO), 0.9% NaCl + 10% methyl glycol, and 0.9% NaCl + 10% dimethyl sulfoxide, loaded into 0.25-mL straws and frozen in liquid nitrogen vapor. The semen from each treatment was thawed at three different thawing rates: 25 C for 30 sec, 30 C for 16 sec and 40 C for 12 sec. Motility, vitality and morphology analyses were performed by computer-assisted sperm analysis (CASA). The characteristics of the fresh sperm mostly resembled those found in the literature. For the parameter...

O Prochilodus brevis é um peixe reofílico, importante componente do ecossistema fluvial e apreciado na culinária nordestina. No entanto, ações antrópicas têm ameaçado sua sobrevivência. Desta forma, surge, nos pesquisadores, o interesse no desenvolvimento de protocolos de conservação do material genético, como a criopreservação seminal. Logo, a determinação do meio de congelação e da taxa de descongelação adequados, são passos fundamentais que possibilitarão a utilização dessa biotecnologia na produção de curimatã comum, reduzindo os riscos à sua sobrevivência. Portanto, o objetivo desse trabalho foi avaliar o efeito de diferentes meios de congelação e taxas de descongelação sobre a qualidade do sêmen criopreservado de P. brevis. Para isso, 18 horas antes da coleta de sêmen, os machos receberam dose única de extrato hipofisário de carpa. Cada animal foi sedado com solução à base de eugenol e o sêmen foi coletado. As amostras foram diluídas em quatro meios de congelação (5% Glicose + Metilglicol 10%; 5% Glicose + DMSO 10%; 0,9% NaCl + Metilglicol 10%; 0,9% NaCl + DMSO 10%) envasadas em palhetas de 0,25 mL e congeladas em vapor de nitrogênio líquido. O sêmen foi descongelado após sete dias em três taxas de descongelação: 25 C 30 s-1; 30 C 16 s-1; 40 C 12 s-1. Foram feitas as análises de motilidade, vitalidade e morfologia com auxílio de sistema automatizado de análise seminal...

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(AU)

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(AU)

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

Presença de antioxidantes no sêmen de teleósteos e sua utilização na suplementação de meios de congelação seminal / Presence of antioxidants in teleost sperm and its use in supplementation of seminal freezing media

A criopreservação seminal é uma ferramenta promissora para o desenvolvimento da aquicultura,entretanto, seus efeitos incluem o aumento da produção de espécies reativas de oxigênio (EROs), provocandodanos à célula espermática e reduzindo sua viabilidade após a descongelação seminal. Além disso, a diluição dosêmen reduz a concentração dos constituintes do plasma seminal, incluindo antioxidantes. Dessa forma, autilização de terapias antioxidantes tem ganhado destaque nos protocolos de criopreservação seminal de peixes.Assim, essa revisão teve como objetivo abordar a utilização de antioxidantes na suplementação de meios decongelação e elencar os antioxidantes empregados com sucesso na criopreservação seminal de peixes teleósteos.A utilização dessas substâncias tem promovido melhorias no movimento espermático, reduzido danos ao DNAespermático e elevado taxas de fertilização e eclosão. Entretanto, sua ação antioxidante sofre elevada variaçãointerespecífica, gerando a necessidade de se testar diferentes tipos de antioxidantes e concentrações, buscando amelhor opção para as espécies de interesse.(AU)

The seminal cryopreservation is a promising tool for the development of aquaculture, however, itseffects include increased production of reactive oxygen species (ROS), causing damage to sperm cells andreducing their viability after thaw seminal. Furthermore, the semen dilution reduces the concentration ofseminal plasma constituents, including antioxidants. The use of antioxidant therapies has been notable in fishseminal cryopreservation protocols. Therefore, this review aimed to address the use of antioxidantssupplementation in freezing media and list the antioxidants employed successfully in seminal cryopreservation ofteleost fish. The use of these substances has improved sperm movement, reduced damage to sperm DNA andincreased fertilization and hatching rates. However, the action of these substances suffer high interspecificvariation, generating the need to test different types of antioxidants and concentrations, seeking the best optionfor the species of interest.(AU)

Dose inseminante e resfriamento de embriões de peixes de água doce / Insemination dose and conservation of freshwater fish embryos

A aquicultura tem sido utilizada como importante ferramenta para suprir o constante aumento doconsumo de pescado. Um dos principais aspectos para a intensificação da produção piscícola, acompanhada dasustentabilidade tanto econômica quanto ambiental, é a utilização de técnicas de propagação artificial. Dessaforma, para otimizar a reprodução artificial têm-se investido em estudos afim de definir a proporção ideal deespermatozoides por ovócito e os melhores métodos de conservação para embriões de espécies de peixes de águadoce. Objetivou-se fazer uma breve revisão sobre as doses inseminantes e a biotecnologia de resfriamento deembriões de peixes de água doce que já foram estabelecidas.(AU)

Aquaculture has been used as an important tool to meet the steady increase in fish consumption. Amajor aspect for the intensification of fish production, accompanied by both economic and environmentalsustainability, is the use of techniques of artificial propagation. Thus, to optimize artificial reproduction havebeen invested in research in order to define the optimal ratio the sperm to oocyte and the best conservationmethods for embryos species of freshwater fish. The objective was to make a brief review of the inseminationdoses cooling biotechnology freshwater fish embryos that have already been established.(AU)

Presença de antioxidantes no sêmen de teleósteos e sua utilização na suplementação de meios de congelação seminal / Presence of antioxidants in teleost sperm and its use in supplementation of seminal freezing media

A criopreservação seminal é uma ferramenta promissora para o desenvolvimento da aquicultura,entretanto, seus efeitos incluem o aumento da produção de espécies reativas de oxigênio (EROs), provocandodanos à célula espermática e reduzindo sua viabilidade após a descongelação seminal. Além disso, a diluição dosêmen reduz a concentração dos constituintes do plasma seminal, incluindo antioxidantes. Dessa forma, autilização de terapias antioxidantes tem ganhado destaque nos protocolos de criopreservação seminal de peixes.Assim, essa revisão teve como objetivo abordar a utilização de antioxidantes na suplementação de meios decongelação e elencar os antioxidantes empregados com sucesso na criopreservação seminal de peixes teleósteos.A utilização dessas substâncias tem promovido melhorias no movimento espermático, reduzido danos ao DNAespermático e elevado taxas de fertilização e eclosão. Entretanto, sua ação antioxidante sofre elevada variaçãointerespecífica, gerando a necessidade de se testar diferentes tipos de antioxidantes e concentrações, buscando amelhor opção para as espécies de interesse.

The seminal cryopreservation is a promising tool for the development of aquaculture, however, itseffects include increased production of reactive oxygen species (ROS), causing damage to sperm cells andreducing their viability after thaw seminal. Furthermore, the semen dilution reduces the concentration ofseminal plasma constituents, including antioxidants. The use of antioxidant therapies has been notable in fishseminal cryopreservation protocols. Therefore, this review aimed to address the use of antioxidantssupplementation in freezing media and list the antioxidants employed successfully in seminal cryopreservation ofteleost fish. The use of these substances has improved sperm movement, reduced damage to sperm DNA andincreased fertilization and hatching rates. However, the action of these substances suffer high interspecificvariation, generating the need to test different types of antioxidants and concentrations, seeking the best optionfor the species of interest.

Dose inseminante e resfriamento de embriões de peixes de água doce / Insemination dose and conservation of freshwater fish embryos

A aquicultura tem sido utilizada como importante ferramenta para suprir o constante aumento doconsumo de pescado. Um dos principais aspectos para a intensificação da produção piscícola, acompanhada dasustentabilidade tanto econômica quanto ambiental, é a utilização de técnicas de propagação artificial. Dessaforma, para otimizar a reprodução artificial têm-se investido em estudos afim de definir a proporção ideal deespermatozoides por ovócito e os melhores métodos de conservação para embriões de espécies de peixes de águadoce. Objetivou-se fazer uma breve revisão sobre as doses inseminantes e a biotecnologia de resfriamento deembriões de peixes de água doce que já foram estabelecidas.

Aquaculture has been used as an important tool to meet the steady increase in fish consumption. Amajor aspect for the intensification of fish production, accompanied by both economic and environmentalsustainability, is the use of techniques of artificial propagation. Thus, to optimize artificial reproduction havebeen invested in research in order to define the optimal ratio the sperm to oocyte and the best conservationmethods for embryos species of freshwater fish. The objective was to make a brief review of the inseminationdoses cooling biotechnology freshwater fish embryos that have already been established.

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.

Sperm motility and velocities of Characiformes fishes in different times post-activation / Motilidade e velocidades espermáticas de peixes Characiformes em diferentes tempos pós ativação

The objective of the current study was to observe the performance kinetics (motilities and velocities) of the spermatozoa from Prochilodus brevis (curimatã), Colossoma macropomum (tambaqui) and Piaractus brachypomus (pirapitinga) species in different times post-activation. The sperm of P. brevis, C. macropomum and P. brachypomus species were collected after hormonal induction with carp pituitary extract. The samples with not contamination with water, urine or feces had motility subjective, morphology, osmolality and concentration analyzed. The samples selected were analyzed with Sperm Class Analyzer. Spermatozoa motility and velocities were captured at 10, 30, 60 and 120 s postactivation. No significant differences in total motility of P. brevis spermatozoa were observed between 10 s and 30 s post-activation. However, significant reduction was observed in 60 s. This reduction was more accentuated after 120 s. The same pattern of spermatozoa motility decline happened for C. macropomum and P. brachypomus. Velocities also followed the same pattern for the three species. There was significant reduction in velocities after 30 s; this reduction was more significant after 60 s. There was no significance difference between 60 s and 120 s post-activation. Sperm of C. macropomum and P. brachypomus show satisfactory sperm quality up to 60 s after activation. On the other hand, sperm of P. brevis up to 120 s after activation. These findings show that the rate of sperm motility in different times post activation is change for each species tested.(AU)

O objetivo do presente trabalho foi investigar o desempenho cinético (motilidade e velocidades espermáticas) de Prochilodus brevis (curimatã), Colossoma macropomum (tambaqui) e Piaractus brachypomus (pirapitinga) em diferentes tempos pós ativação. O sêmen de P. brevis, C. macropomum e P. brachypomus foi coletado após indução hormonal com extrato hipofisário de carpa. As amostras não contaminadas com sangue, fezes ou urina tiveram motilidade subjetiva, morfologia, osmolaridade e concentração analisadas. As amostras selecionadas foram analisadas com o Sperm Class Analyzer. A motilidade e velocidade dos espermatozoides foram mensuradas a 10, 30, 60 e 120 s pós ativação. Nenhuma diferença significativa na motilidade total do sêmen de P. brevis foi observada entre 10 s e 30 s pós ativação. No entanto, uma redução significativa foi observada aos 60 s. Essa redução foi mais acentuada após 120 s. O mesmo padrão de declínio da motilidade espermática ocorreu para C. macropomum e P. brachypomus. As velocidades espermáticas seguiram o mesmo padrão para as três espécies. Houve redução significativa nas velocidades após 30 s; essa redução foi mais significativa após 60 s. Não houve diferença significativa entre 60 s e 120 s pós ativação. Os espermatozoides de C. macropomum e P. brachypomus apresentam satisfatotia qualidade espermática até 60 s pós ativação. Por outro lado, os espermatozoides de P. brevis apresentam satisfatória qualidade espermática até 120 s pós ativação. Esses achados mostram que a taxa de motilidade espermática em diferentes tempos pós ativação muda para cada espécie testada.(AU)

Conservação de gametas e embriões de peixes teleósteos / Preservation of gametes and embryo of teleost fish

Objetivou-se com este manuscrito realizar uma breve revisão sobre técnicas de reprodução assistida utilizadas para peixes, apresentando uma visão geral sobre a conservação de gametas e embriões e os avanços tecnológicos já alcançados. Contextualizando com sua importância para a preservação de material genética e aplicação comercial.(AU)

The aim of this manuscript was to conduct a brief review about assisted reproduction techniques used to fish, presenting an overview of the gametes and embryos conservation and technological advances already achieved in this field. Contextualizing to their importance for the preservation of genetic material and commercial application.(AU)