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)

Supplementation of cryodiluent medium with sulfated polysaccharides from green seaweeds in the freezing of Colossoma macropomum semen / Suplementação do meio criodiluidor com polissacarídeos sulfatados de macroalgas verdes na congelação do sêmen de Colossoma macropomum

This study proposes to investigate the addition of sulfated polysaccharides (SP) extracted from two species of green seaweeds, Ulva lactuca and Caulerpa racemosa, to Colossoma macropomum semen cryodiluent medium. Four concentrations of SP (1.0, 2.0, 3.0, or 4.0 mg mL-1) of each seaweed were evaluated. Semen was collected during the month of September in Fortaleza - CE, Brazil. Fresh semen samples were analyzed for the parameters of total sperm motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), sperm morphology, membrane integrity, and DNA integrity. Then, the samples were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO) + 5% glucose, which was supplemented with different concentrations of SP. An unsupplemented treatment was used as control. After 15 days, they were thawed in a water bath at 45 ºC for eight seconds and the same analyses of fresh semen were performed. Statistical analysis revealed that there were no significant differences (p > 0.05) between the different tested concentrations of SP for any of the evaluated parameters. Compared with the control, there was no difference in concentrations (p > 0.05) for total motility; however, for VCL, VSL, and VAP, the U. lactuca concentrations of 3.0 and 4.0 mg mL-1 were detrimental (p < 0.05). The same was observed with 4.0 mg mL-1 of C. racemosa for VSL and VAP. In terms of morphology, 1.0 and 4.0 mg mL-1 of C. racemosa reduced normal sperm (p < 0.05), whereas for the other concentrations there was no difference (p > 0.05). All concentrations of both seaweeds maintained plasma membrane integrity (p > 0.05). As for DNA integrity, only 4.0 mg mL-1 of U. lactuca produced lower results than the control (p < 0.05), whereas the other concentrations maintained the number of spermatozoa with intact DNA (p > 0.05). Based on the results, higher concentrations of SP are harmful to tambaqui sperm in the freezing medium, whereas lower concentrations maintain sperm parameters. Further research is warranted to better investigate the antioxidant potential of these polymers in cryodiluent medium for C. macropomum as well as other fish species.

O estudo teve como objetivo avaliar a adição de polissacarídeos sulfatados (PS) extraídos de duas espécies de macroalgas verdes, Ulva lactuca e Caulerpa racemosa, no meio criodiluidor do sêmen de Colossoma macropomum. Para isso, foram avaliadas quatro concentrações de PS (1,0; 2,0; 3,0 ou 4,0 mg mL-1), de cada macroalga. A coleta de sêmen foi realizada durante o mês de setembro, em Fortaleza, Ceará, Brasil. As amostras de sêmen fresco foram analisadas quanto aos parâmetros de motilidade total dos espermatozoides, velocidade curvilinear (VCL), velocidade em linha reta (VSL), velocidade média do trajeto (VAP), morfologia espermática, integridade de membrana e integridade de DNA. Em seguida, foram criopreservadas em meio de congelação contendo dimetilsulfóxido (DMSO) 10% + glicose 5%, e suplementadas com as diferentes concentrações de PS, tendo ainda um tratamento não suplementado como controle. Após 15 dias, foram descongeladas em banho-maria a 45 ºC por oito segundos, e as mesmas análises do sêmen fresco foram realizadas. Através da análise estatística, os resultados mostraram que não houveram diferenças significativas (p > 0,05) entre as diferentes concentrações de PS testadas para nenhum dos parâmetros avaliados. Já em relação ao controle, não houve diferença nas concentrações (p > 0,05) para a motilidade total, no entanto, para VCL, VSL e VAP, as concentrações de 3,0 e 4,0 mg mL-1 de U. lactuca foram prejudiciais (p < 0,05). O mesmo foi observado em 4,0 mg mL-1 de C. racemosa para VSL e VAP. Para a morfologia, 1,0 e 4,0 mg mL-1 de C. racemosa reduziram os espermatozoides normais (p < 0,05), enquanto para as demais concentrações não houve diferença (p > 0,05). Para todas as concentrações de ambas as macroalgas, a integridade de membrana plasmática foi mantida (p > 0,05). Quanto à integridade do DNA, apenas 4,0 mg mL-1 de U. lactuca foi inferior ao controle (p < 0,05), enquanto as demais concentrações mantiveram o número de espermatozoides com DNA íntegro (p > 0,05). De acordo com os resultados obtidos, concentrações mais elevadas de PS são prejudiciais aos espermatozoides de tambaqui no meio de congelação, enquanto concentrações mais baixas mantiveram parâmetros espermáticos. Estudos posteriores são indicados para melhor avaliar o potencial antioxidante destes polímeros no meio criodiluidor do sêmen de C. macropomum, bem como de outras espécies de peixes.

Alimentação de pós-larvas de teleósteos e sua importância na produção em cativeiro / Feeding of teleost larvae and their importance in captive production

A alimentação de pós-larvas de peixes é um dos momentos mais difíceis da produção em cativeiro, pois os animais iniciam uma alimentação exógena e apresentam um sistema digestório pouco desenvolvido. Com isso, é sugerida a oferta de alimento vivo (plâncton), uma vez que são fonte de nutrientes essenciais e também porque é comprovado o aumento das taxas de sobrevivência e crescimento para algumas espécies de pós-larvas de peixes. Os plânctons mais ofertados são os náuplios de Artemia nauplii, copepod e rotifers. Objetivou-se descrever os entraves que envolvem a nutrição de larvas de peixes, desde o momento em que o animal abre a boca, e os tipos de alimentos ideais para o seu melhor crescimento e sobrevivência.

Feeding fish post-larvae is one of the most difficult moments of production in captivity, as animals start an exogenous diet and have a poorly developed digestive system. Thus, the supply of live food (plankton) is suggested, since they are a source of essential nutrients and also because it has been proven to increase survival and growth rates for some species of fish post-larvae. The most frequently offered plankton are the Artemia nauplii, copepod and rotifers. The objective was to describe the obstacles that involve the nutrition of fish larvae, from the moment the animal opens its mouth, and the ideal types of feed for its best growth and survival.

Determinação e diferenciação sexual em peixes teleósteos: papel dos fatores ambientais e genéticos / Sexual determination and differentiation in teleost fish: the role of environmental and genetic factors

Peixes teleósteos possuem diversas maneiras de determinação e diferenciação sexual. De modo que, fatores genéticos e ambientais podem atuar interagindo para determinar o sexo e agir sobre a diferenciação das gônadas desses animais. Os fatores ambientais atuam de diferentes formas para determinar o sexo de peixe, uma vez que sua ação é dependente da espécie. Fatores como temperatura, pH, densidade populacional e poluição podem causara masculinização ou feminização, agindo principalmente sobre os hormônios sexuais e muitas vezes causando a apoptose ovariana e, consequentemente, promovendo a masculinização. Já a determinação genética se dar por cromossomos sexuais (sistema monogênico) ou por ação autossômica (sistema poligênica). No sistema monogênico já foram observados vários modelos de cromossomos sexuais, sendo os principais XX/XY (machos heterogaméticos) e ZW/ZZ (fêmeas heterogaméticas). Com isso, é possível observar que os peixes teleósteos possuem uma grande diversidade de determinação sexual e diferenciação gonadal.

Teleost fish have various forms of sexual determination and differentiation. Genetic and environmental factors can interact to determine sex and act on the differentiation of the gonads of these animals. Environmental factors act in different ways to determine the sex of the fish, as their action depend on the species. Factors such as temperature, pH, population density and pollution can cause a masculinization or a feminization, by acting on the hormones and, often, the ovarian apoptosis. The genetic determination is given by the sexual chromosome (monogenic system) or by the autosomal action (polygenic system). In the monogenic system several models of sexual chromosomes were observed, the main ones being XX / XY (heterogeneous males) and ZW / ZZ(heterogeneous females). With this, it is possible to observe that teleost possess a great amplitude of sexual determination and gonadal differentiation.

Determinação e diferenciação sexual em peixes teleósteos: papel dos fatores ambientais e genéticos / Sexual determination and differentiation in teleost fish: the role of environmental and genetic factors

Peixes teleósteos possuem diversas maneiras de determinação e diferenciação sexual. De modo que, fatores genéticos e ambientais podem atuar interagindo para determinar o sexo e agir sobre a diferenciação das gônadas desses animais. Os fatores ambientais atuam de diferentes formas para determinar o sexo de peixe, uma vez que sua ação é dependente da espécie. Fatores como temperatura, pH, densidade populacional e poluição podem causara masculinização ou feminização, agindo principalmente sobre os hormônios sexuais e muitas vezes causando a apoptose ovariana e, consequentemente, promovendo a masculinização. Já a determinação genética se dar por cromossomos sexuais (sistema monogênico) ou por ação autossômica (sistema poligênica). No sistema monogênico já foram observados vários modelos de cromossomos sexuais, sendo os principais XX/XY (machos heterogaméticos) e ZW/ZZ (fêmeas heterogaméticas). Com isso, é possível observar que os peixes teleósteos possuem uma grande diversidade de determinação sexual e diferenciação gonadal.(AU)

Teleost fish have various forms of sexual determination and differentiation. Genetic and environmental factors can interact to determine sex and act on the differentiation of the gonads of these animals. Environmental factors act in different ways to determine the sex of the fish, as their action depend on the species. Factors such as temperature, pH, population density and pollution can cause a masculinization or a feminization, by acting on the hormones and, often, the ovarian apoptosis. The genetic determination is given by the sexual chromosome (monogenic system) or by the autosomal action (polygenic system). In the monogenic system several models of sexual chromosomes were observed, the main ones being XX / XY (heterogeneous males) and ZW / ZZ(heterogeneous females). With this, it is possible to observe that teleost possess a great amplitude of sexual determination and gonadal differentiation.(AU)

Alimentação de pós-larvas de teleósteos e sua importância na produção em cativeiro / Feeding of teleost larvae and their importance in captive production

A alimentação de pós-larvas de peixes é um dos momentos mais difíceis da produção em cativeiro, pois os animais iniciam uma alimentação exógena e apresentam um sistema digestório pouco desenvolvido. Com isso, é sugerida a oferta de alimento vivo (plâncton), uma vez que são fonte de nutrientes essenciais e também porque é comprovado o aumento das taxas de sobrevivência e crescimento para algumas espécies de pós-larvas de peixes. Os plânctons mais ofertados são os náuplios de Artemia nauplii, copepod e rotifers. Objetivou-se descrever os entraves que envolvem a nutrição de larvas de peixes, desde o momento em que o animal abre a boca, e os tipos de alimentos ideais para o seu melhor crescimento e sobrevivência.(AU)

Feeding fish post-larvae is one of the most difficult moments of production in captivity, as animals start an exogenous diet and have a poorly developed digestive system. Thus, the supply of live food (plankton) is suggested, since they are a source of essential nutrients and also because it has been proven to increase survival and growth rates for some species of fish post-larvae. The most frequently offered plankton are the Artemia nauplii, copepod and rotifers. The objective was to describe the obstacles that involve the nutrition of fish larvae, from the moment the animal opens its mouth, and the ideal types of feed for its best growth and survival.(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.

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)

Utilização de proteínas anticongelantes na criopreservação seminal de peixes / Use of anti-freezing proteins in seminal fish cryopreservation

A criopreservação seminal é uma das biotécnica reprodutivas mais promissoras para a aquicultura, pois permite o transporte de gametas, garante melhorias genéticas em espécies desejadas e minimiza a assincronia entre machos e fêmeas reprodutores. Apesar dos benefícios, danos celulares podem ser ocasionados durante o processo de congelação, causando a diminuição da qualidade dos espermatozoides. Sendo assim, estudos que busquem alternativas que minimizem tais danos são de grande importância. As proteínas anticongelantes (AFPs) são importantes compostos originados a partir de processos evolutivos em organismos que vivem em ambientes polares. As AFPs podem ser utilizadas como possíveis agentes crioprotetores, pois são capazes de diminuir os cristais de gelo e proteger a membrana plasmática das células durante o processo de congelação. Dessa forma, esta revisão tem como objetivo reunir informações acerca da utilização de proteínas anticongelantes na conservação do sêmen de peixes e apresentar os principais resultados encontrados na literatura. Observou-se que a utilização dessas substâncias na criopreservação seminal em peixes promoveu melhorias nas taxas de motilidade espermática, velocidades e integridade de membrana plasmática. Tais melhorias foram observadas de maneira interespecífica. Portanto, ainda são necessários estudos mais aprofundados com intuito de verificar o efeito das AFPs na capacidade de fertilização, buscando a melhor opção para suplementação do meio.

Seminal cryopreservation is one of the most promising reproductive biotechniques for aquaculture, as it allows the transport of gametes, ensures genetic improvements in desired species and minimizes asynchrony between breeding males and females. Despite the benefits, cell damage can be caused during the freezing process, causing decreased quality of sperm. Therefore, studies that seek alternatives that minimize such damage are of great importance. Antifreeze proteins (AFPs) are important compounds originated from evolutionary processes in organisms that live in polar environments. AFPs can be used as possible cryoprotective agents, as they are able to decrease ice crystals and protect the plasma membrane of cells during the freezing process. Thus, this review aims to gather information about the use of antifreeze proteins in the conservation of fish semen and present the main results found in the literature. It was observed that the use of these substances in seminal cryopreservation in fish promoted improvements in the rates of sperm motility, velocities and integrity of the plasma membrane. Such improvements were observed in an interspecific manner. Therefore, further studies are needed in order to verify the effect of AFPs on fertilization capacity, seeking the best option for supplementing the medium.

Utilização de proteínas anticongelantes na criopreservação seminal de peixes / Use of anti-freezing proteins in seminal fish cryopreservation

A criopreservação seminal é uma das biotécnica reprodutivas mais promissoras para a aquicultura, pois permite o transporte de gametas, garante melhorias genéticas em espécies desejadas e minimiza a assincronia entre machos e fêmeas reprodutores. Apesar dos benefícios, danos celulares podem ser ocasionados durante o processo de congelação, causando a diminuição da qualidade dos espermatozoides. Sendo assim, estudos que busquem alternativas que minimizem tais danos são de grande importância. As proteínas anticongelantes (AFPs) são importantes compostos originados a partir de processos evolutivos em organismos que vivem em ambientes polares. As AFPs podem ser utilizadas como possíveis agentes crioprotetores, pois são capazes de diminuir os cristais de gelo e proteger a membrana plasmática das células durante o processo de congelação. Dessa forma, esta revisão tem como objetivo reunir informações acerca da utilização de proteínas anticongelantes na conservação do sêmen de peixes e apresentar os principais resultados encontrados na literatura. Observou-se que a utilização dessas substâncias na criopreservação seminal em peixes promoveu melhorias nas taxas de motilidade espermática, velocidades e integridade de membrana plasmática. Tais melhorias foram observadas de maneira interespecífica. Portanto, ainda são necessários estudos mais aprofundados com intuito de verificar o efeito das AFPs na capacidade de fertilização, buscando a melhor opção para suplementação do meio.(AU)

Seminal cryopreservation is one of the most promising reproductive biotechniques for aquaculture, as it allows the transport of gametes, ensures genetic improvements in desired species and minimizes asynchrony between breeding males and females. Despite the benefits, cell damage can be caused during the freezing process, causing decreased quality of sperm. Therefore, studies that seek alternatives that minimize such damage are of great importance. Antifreeze proteins (AFPs) are important compounds originated from evolutionary processes in organisms that live in polar environments. AFPs can be used as possible cryoprotective agents, as they are able to decrease ice crystals and protect the plasma membrane of cells during the freezing process. Thus, this review aims to gather information about the use of antifreeze proteins in the conservation of fish semen and present the main results found in the literature. It was observed that the use of these substances in seminal cryopreservation in fish promoted improvements in the rates of sperm motility, velocities and integrity of the plasma membrane. Such improvements were observed in an interspecific manner. Therefore, further studies are needed in order to verify the effect of AFPs on fertilization capacity, seeking the best option for supplementing the 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.

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)

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)

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.

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

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

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

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

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...

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)

Perfil proteico do plasma seminal ovino e sua relação com a criopreservação nos diluentes TRIS e Água de Coco em Pó / Protein profile of ram seminal plasm and its relation relationship with cryopreservation in TRIS and Powdered Coconut Water extender

O objetivo deste trabalho foi avaliar a relação entre a concentração de proteínas no plasma seminal ovino com parâmetros de qualidade do sêmen criopreservado em diluentes TRIS e em Água de Coco em Pó (ACP102c). Um total de 12 colheitas/animal em ovinos Santa Inês (n=2) e Dorper (n=2) foram realizadas, para obtenção do plasma seminal e para criopreservação nos diluentes testados. Foi utilizado o método de Bradford para determinação da concentração de proteína. Para avaliação do sêmen fresco e criopreservado foram utilizados os testes de viabilidade por eosina-nigrosina e teste hiposmótico (HOST), além dos parâmetros de motilidade em sistema de análise de sêmen auxiliada por computador (CASA). Baseado no valor médio da concentração de proteína, os ejaculados foram agrupados em alta (AP) e baixa (BP) concentração proteica. Correlações significativamente positivas foram encontradas entre a concentração de proteína com os parâmetros motilidade progressiva (PROG) e congelabilidade (CONGEL) e negativa para deslocamento lateral de cabeça (ALH) nas amostras criopreservadas em ambos diluentes, além de correlação positiva para motilidade total (MOT) em TRIS. Sêmen de ejaculados do grupo AP criopreservados em TRIS apresentaram MOT, PROG e CONGEL significativamente superiores ao grupo BP e inferiores para ALH. Quando criopreservado em ACP102c, sêmen do grupo AP foi superior ao BP para PROG e CONGEL. Variações na qualidade do sêmen criopreservado entre ejaculados podem estar associados à concentração de proteínas do plasma seminal, e estas podem atuar na manutenção da progressividade de espermatozoides ovinos criopreservados em diluentes TRIS e ACP102c.(AU)

The objective of this study was to evaluate the relation between the protein concentration in ram seminal plasma and quality parameters of the semen cryopreserved in TRIS and Powdered Coconut Water (ACP102c) extenders. A total of 12 semen collections/animal of Santa Ines (n=2) and Dorper (n=2) rams were performed to obtain seminal plasma and semen cryopreservation in work extenders. The protein content was determined using the Bradford's method. The eosin-nigrosine and hyposmotic (HOST) viable tests and motility parameters obtained by computer assisted semen analysis were evaluated for fresh and cryopreserved semen. The ejaculates were grouped in high (AP) and low (BP) protein content, based on the mean value of protein concentration. Significantly positive correlations were found between the protein content and progressive motility (PROG), freezability (CONGEL) and negative for lateral head displacement (ALH) in the semen cryopreserved in both extenders. Therefore, positive correlation was found for total motility (MOT) in TRIS. Semen samples from AP group cryopreserved in TRIS extender were significantly better than BP group for MOT, PROG and CONGEL, and inferior for ALH. When cryopreserved in ACP102c, the AP group were better than BP group for PROG and CONGEL. Variations in the quality of cryopreserved semen from different ejaculates may be associated with the concentration of seminal plasma proteins and may act to maintain the progressiveness of ram sperm cryopreserved in TRIS and ACP102c extenders.(AU)