Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants

Abstract The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me2SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me2SO.

Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants

The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me2SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me2SO.(AU)

Use of glycosaminoglycans from Oreochromisniloticus skin as an antioxidant supplement for milt cryopreservation of Brazilian bocachico / Uso de glicosaminoglicanos da pele Oreochromis niloticus como suplemento antioxidante na criopreservação de sêmen de curimatã comum (Prochilodus brevis)

The study aimed to evaluate the in vitro antioxidant action of glycosaminoglycans (GAGs) from the skinof Oreochromis niloticus, and to determine their ideal concentration to supplement the sperm freezing medium of Prochilodus brevis. In experiment 1, the in vitro antioxidant properties of GAGs were verified through the analysis of DPPH, chelating ferrous ability, and total antioxidant capacity. In experiment 2, milt pools were formed, which were frozen in solution supplemented or not with different GAGs concentrations: 0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg mL-¹ (total of 10 treatments). The samples were evaluated for membrane integrity, DNA integrity, sperm morphology, and sperm kinetics. The results of experiment 1 showed that the GAGs exhibited, with the increase of the concentration, significant antioxidant action, for all the evaluated tests, mainly in the chelating ferrous ability. In experiment 2, it was observed that the increase of GAGs concentration decreased kinetic parameters (P < 0.05), however, the control and 0.5 mg mL-1 GAGs concentration showed similar results. For the other parameters (membrane integrity, DNA integrity, and sperm morphology), there was no decrease in results with the increase of GAGs concentration. In conclusion, GAGs extracted from O. niloticus skin have antioxidant action, and the concentration of 0.5 mg mL-¹ was the most adequate to supplement the P. brevis sperm-freezing medium.(AU)

O estudo teve como objetivo avaliar in vitro a ação antioxidante de glicosaminoglicanos (GAGs) da pele de Oreochromis niloticus e determinar sua concentração ideal para suplementar o meio de congelação espermático de Prochilodus brevis. No experimento 1, foram verificadas as propriedades antioxidantes in vitro dos GAGs por meio das análises de DPPH, capacidade quelante do ferro e capacidade antioxidante total. No experimento 2, foram formados pool de sêmen, que foram congelados em solução suplementada, ou não, com diferentes concentrações de GAGs: 0 (controle); 0,5; 1,0; 1,5; 2,0; 2,5; 3,0; 3,5; 4,0; 4,5 ou5,0 mg mL-¹ (total de 10 tratamentos). As amostras foram avaliadas quanto à integridade da membrana, integridade do DNA, morfologia e cinética espermática. Os resultados do experimento 1, mostraram que os GAGs exibiram, com o aumento da concentração, ação antioxidante significativa, para todos os testes avaliados, principalmente na capacidade quelante do ferro. No experimento 2, observou-se que o aumento da concentração de GAGs diminuiu os parâmetros cinéticos (P < 0,05), porém o controle e a concentração de 0,5 mg mL-1 de GAGs apresentaram resultados semelhantes. Para os demais parâmetros (morfologia, integridade de membrana e de DNA), não houve diminuição dos resultados com o aumento da concentração de GAGs. Em conclusão, os GAGs, extraídos da pele de O. niloticus, possuem ação antioxidante, sendo a concentração de 0,5 mg mL-1 a mais adequada para suplementar o meio de congelação espermático de P. brevis.(AU)

Use of glycosaminoglycans from Oreochromisniloticus skin as an antioxidant supplement for milt cryopreservation of Brazilian bocachico / Uso de glicosaminoglicanos da pele Oreochromis niloticus como suplemento antioxidante na criopreservação de sêmen de curimatã comum (Prochilodus brevis)

The study aimed to evaluate the in vitro antioxidant action of glycosaminoglycans (GAGs) from the skinof Oreochromis niloticus, and to determine their ideal concentration to supplement the sperm freezing medium of Prochilodus brevis. In experiment 1, the in vitro antioxidant properties of GAGs were verified through the analysis of DPPH, chelating ferrous ability, and total antioxidant capacity. In experiment 2, milt pools were formed, which were frozen in solution supplemented or not with different GAGs concentrations: 0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg mL-¹ (total of 10 treatments). The samples were evaluated for membrane integrity, DNA integrity, sperm morphology, and sperm kinetics. The results of experiment 1 showed that the GAGs exhibited, with the increase of the concentration, significant antioxidant action, for all the evaluated tests, mainly in the chelating ferrous ability. In experiment 2, it was observed that the increase of GAGs concentration decreased kinetic parameters (P < 0.05), however, the control and 0.5 mg mL-1 GAGs concentration showed similar results. For the other parameters (membrane integrity, DNA integrity, and sperm morphology), there was no decrease in results with the increase of GAGs concentration. In conclusion, GAGs extracted from O. niloticus skin have antioxidant action, and the concentration of 0.5 mg mL-¹ was the most adequate to supplement the P. brevis sperm-freezing medium.

O estudo teve como objetivo avaliar in vitro a ação antioxidante de glicosaminoglicanos (GAGs) da pele de Oreochromis niloticus e determinar sua concentração ideal para suplementar o meio de congelação espermático de Prochilodus brevis. No experimento 1, foram verificadas as propriedades antioxidantes in vitro dos GAGs por meio das análises de DPPH, capacidade quelante do ferro e capacidade antioxidante total. No experimento 2, foram formados pool de sêmen, que foram congelados em solução suplementada, ou não, com diferentes concentrações de GAGs: 0 (controle); 0,5; 1,0; 1,5; 2,0; 2,5; 3,0; 3,5; 4,0; 4,5 ou5,0 mg mL-¹ (total de 10 tratamentos). As amostras foram avaliadas quanto à integridade da membrana, integridade do DNA, morfologia e cinética espermática. Os resultados do experimento 1, mostraram que os GAGs exibiram, com o aumento da concentração, ação antioxidante significativa, para todos os testes avaliados, principalmente na capacidade quelante do ferro. No experimento 2, observou-se que o aumento da concentração de GAGs diminuiu os parâmetros cinéticos (P < 0,05), porém o controle e a concentração de 0,5 mg mL-1 de GAGs apresentaram resultados semelhantes. Para os demais parâmetros (morfologia, integridade de membrana e de DNA), não houve diminuição dos resultados com o aumento da concentração de GAGs. Em conclusão, os GAGs, extraídos da pele de O. niloticus, possuem ação antioxidante, sendo a concentração de 0,5 mg mL-1 a mais adequada para suplementar o meio de congelação espermático de P. brevis.

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.

Vitrificação de sêmen de peixes teleósteos / Vitrification of teleosts fish semen

A preservação de sêmen de peixes é uma técnica promissora para o desenvolvimento daaquicultura, e consiste na conservação dos gametas masculinos a longo prazo. Dentre os procedimentosde criopreservação de sêmen de peixes, a vitrificação vem sendo desenvolvida como uma alternativa àcriopreservação convencional devido à sua rapidez, praticidade e baixo custo. Apesar dos benefícios, essatécnica possui alguns entraves que limitam seu sucesso, uma vez que consiste em mergulhar a amostradiretamente em nitrogênio líquido, necessitando de grandes quantidades de crioprotetores de ação interna,que podem ser tóxicos. Dessa forma, diferentes protocolos precisam ser testados, a fim de obterresultados satisfatórios após a aplicação da técnica. A vitrificação tem sido utilizada com sucesso parasêmen, ovócitos, embriões e tecidos de mamíferos e, atualmente, alguns protocolos de vitrificação desêmen já foram elaborados para determinadas espécies de peixes com relativo sucesso, contudo maisestudos são necessários para aumentar a viabilidade espermática após o reaquecimento. Portanto, oobjetivo desta revisão é resumir os procedimentos básicos do processo de vitrificação de sêmen de peixese apresentar os principais resultados encontrados na literatura.(AU)

The preservation of fish semen is a promising technique for the development of aquaculture, andconsists in the conservation of male gametes in the long term. Among the procedures forcryopreservation of fish semen, vitrification has been developed as an alternative to conventionalcryopreservation due to its speed, practicality, and low cost. Despite the benefits, this technique has someobstacles that limit its success, since it consists of immersing the sample directly into liquid nitrogen,requiring large amounts of internal cryoprotectants, which can be toxic. Thus, different protocols need tobe tested in order to obtain satisfactory results after the application of the technique. Vitrification hasbeen successfully used for semen, oocytes, embryos and mammalian tissues, and currently some semenvitrification protocols have been developed for certain species of fish with relative success, howeverfurther studies are needed to increase sperm viability after warming. Therefore, the purpose of thisreview is to summarize the basic procedures of the fish semen vitrification process and present the mainresults found in the literature.(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.(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.(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.

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.

Vitrificação de sêmen de peixes teleósteos / Vitrification of teleosts fish semen

A preservação de sêmen de peixes é uma técnica promissora para o desenvolvimento daaquicultura, e consiste na conservação dos gametas masculinos a longo prazo. Dentre os procedimentosde criopreservação de sêmen de peixes, a vitrificação vem sendo desenvolvida como uma alternativa àcriopreservação convencional devido à sua rapidez, praticidade e baixo custo. Apesar dos benefícios, essatécnica possui alguns entraves que limitam seu sucesso, uma vez que consiste em mergulhar a amostradiretamente em nitrogênio líquido, necessitando de grandes quantidades de crioprotetores de ação interna,que podem ser tóxicos. Dessa forma, diferentes protocolos precisam ser testados, a fim de obterresultados satisfatórios após a aplicação da técnica. A vitrificação tem sido utilizada com sucesso parasêmen, ovócitos, embriões e tecidos de mamíferos e, atualmente, alguns protocolos de vitrificação desêmen já foram elaborados para determinadas espécies de peixes com relativo sucesso, contudo maisestudos são necessários para aumentar a viabilidade espermática após o reaquecimento. Portanto, oobjetivo desta revisão é resumir os procedimentos básicos do processo de vitrificação de sêmen de peixese apresentar os principais resultados encontrados na literatura.

The preservation of fish semen is a promising technique for the development of aquaculture, andconsists in the conservation of male gametes in the long term. Among the procedures forcryopreservation of fish semen, vitrification has been developed as an alternative to conventionalcryopreservation due to its speed, practicality, and low cost. Despite the benefits, this technique has someobstacles that limit its success, since it consists of immersing the sample directly into liquid nitrogen,requiring large amounts of internal cryoprotectants, which can be toxic. Thus, different protocols need tobe tested in order to obtain satisfactory results after the application of the technique. Vitrification hasbeen successfully used for semen, oocytes, embryos and mammalian tissues, and currently some semenvitrification protocols have been developed for certain species of fish with relative success, howeverfurther studies are needed to increase sperm viability after warming. Therefore, the purpose of thisreview is to summarize the basic procedures of the fish semen vitrification process and present the mainresults found in the literature.

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)

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...

Dose inseminante e embriogênese na fertilização assistida de carpa comum no nordeste brasileiro / Inseminating dose and embryogenesis in assisted fertilization of common carp in northeastern of Brazil

A presente pesquisa visou determinar a dose inseminante para a fertilização assistida da carpa comum, e o registro em imagens e o tempo de desenvolvimento de cada fase embrionária desta espécie em latitude equatorial. Verificou-se que a porcentagem de fertilização aumentou de forma linear, atingindo um platô em 45,5% na proporção de 208.295 espermatozoides/oócito. Assim, recomenda-se o uso da dose inseminante de aproximadamente 200.000 espermatozoides/oócito na rotina de fertilização assistida dessa espécie, no nordeste brasileiro. Além disso, o desenvolvimento embrionário da carpa em latitude equatorial segue a cronologia semelhante ao relatado para a espécie em clima temperado.(AU)

The present research aimed at determining insemination dose for artificial fertilization of common carp, and record images and time in the development of each embryonic stage of the species in equatorial latitude. The fertilization rate increased linearly up to the proportion of 208.295 spermatozoa/oocyte, and, from this point, the fertilization rate was maintained at 45.5%. Therefore, we recommend the use of the insemination dose of approximately 200.000 sperm/oocyte in the artificial fertilization routine of common carpin brazilian northeast. Moreover, embryonic development of carp in equatorial latitude follows the chronology similar to that reported for the species in temperate zones.(AU)

Dose inseminante e embriogênese na fertilização assistida de carpa comum no nordeste brasileiro / Inseminating dose and embryogenesis in assisted fertilization of common carp in northeastern of Brazil

A presente pesquisa visou determinar a dose inseminante para a fertilização assistida da carpa comum, e o registro em imagens e o tempo de desenvolvimento de cada fase embrionária desta espécie em latitude equatorial. Verificou-se que a porcentagem de fertilização aumentou de forma linear, atingindo um platô em 45,5% na proporção de 208.295 espermatozoides/oócito. Assim, recomenda-se o uso da dose inseminante de aproximadamente 200.000 espermatozoides/oócito na rotina de fertilização assistida dessa espécie, no nordeste brasileiro. Além disso, o desenvolvimento embrionário da carpa em latitude equatorial segue a cronologia semelhante ao relatado para a espécie em clima temperado.

The present research aimed at determining insemination dose for artificial fertilization of common carp, and record images and time in the development of each embryonic stage of the species in equatorial latitude. The fertilization rate increased linearly up to the proportion of 208.295 spermatozoa/oocyte, and, from this point, the fertilization rate was maintained at 45.5%. Therefore, we recommend the use of the insemination dose of approximately 200.000 sperm/oocyte in the artificial fertilization routine of common carpin brazilian northeast. Moreover, embryonic development of carp in equatorial latitude follows the chronology similar to that reported for the species in temperate zones.

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.[...]

Tecnologia de conservação de sêmen de peixes: resfriamento, congelação e uso de antioxidantes / Semen conservation technology fish: cooling, freezing and use of antioxidantes

Esta revisão tem o intuito de abordar as principais biotécnicas reprodutivas aplicadas à conservaçãoseminal de peixes teleósteos, destacando-se o resfriamento (conservação a curto prazo) e a congelação(conservação a longo prazo). Apesar dos inúmeros benefícios, como a conservação de espécies, sabe-se queessas técnicas podem ocasionar danos às células espermáticas. Dessa forma, a fim de manter a viabilidadecelular durante o resfriamento e após a descongelação, utiliza-se soluções diluidoras adequadas e busca-sesuplementá-las para obter melhores resultados. Portanto, o aprimoramento dessas técnicas torna-se de granderelevância para o desenvolvimento da piscicultura.(AU)

This review aims to address key reproductive biotechnologies applied to the seminal conservationteleost fish, cooling highlighting (short term storage) and freezing (long term storage). Despite the manybenefits, such as species conservation, it is known that these techniques can cause damage to sperm cells. Thus,in order to maintain cell viability during cooling and after thawing, use is made appropriate diluting solutionsand seeks to supplement them for best results. Therefore, the improvement of these techniques becomes of greatimportance for the development of fish farming.(AU)

Tecnologia de conservação de sêmen de peixes: resfriamento, congelação e uso de antioxidantes / Semen conservation technology fish: cooling, freezing and use of antioxidantes

Esta revisão tem o intuito de abordar as principais biotécnicas reprodutivas aplicadas à conservaçãoseminal de peixes teleósteos, destacando-se o resfriamento (conservação a curto prazo) e a congelação(conservação a longo prazo). Apesar dos inúmeros benefícios, como a conservação de espécies, sabe-se queessas técnicas podem ocasionar danos às células espermáticas. Dessa forma, a fim de manter a viabilidadecelular durante o resfriamento e após a descongelação, utiliza-se soluções diluidoras adequadas e busca-sesuplementá-las para obter melhores resultados. Portanto, o aprimoramento dessas técnicas torna-se de granderelevância para o desenvolvimento da piscicultura.

This review aims to address key reproductive biotechnologies applied to the seminal conservationteleost fish, cooling highlighting (short term storage) and freezing (long term storage). Despite the manybenefits, such as species conservation, it is known that these techniques can cause damage to sperm cells. Thus,in order to maintain cell viability during cooling and after thawing, use is made appropriate diluting solutionsand seeks to supplement them for best results. Therefore, the improvement of these techniques becomes of greatimportance for the development of fish farming.

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)