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.

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.[...]

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

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.

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)

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.

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.