Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol

This study aimed to determine the semen characteristics of Astyanax lacustris after hormonal induction and to evaluate the sensitivity of the species sperm to cryoprotective solutions based on the cryoprotectants dimethyl sulfoxide and methyl glycol. Volume, color, sperm concentration, total motility and aspects of sperm movement were analyzed using "Integrated Semen Analysis System". Three different extenders were tested: A) glucose 5%+egg yolk 10%, B) BTS®5% and C) glucose 5% and two permeable cryoprotectants: dimethyl sulfoxide (Me2SO) and methyl glycol (MTG). Fresh A. lacustris semen presented total motility of 76.6±11.2%, motility duration of 33.0±2.2s, sperm concentration of 7.22±3.2×109sptz/mL and seminal osmolality of 219±0.03mOsm/kg-1. The toxicity test showed the highest total motility values at the MTG15%+A, Me2SO15%+B and Me2SO10%+C dilutions, and the Me2SO10%+C and Me2SO15%+C dilutions presented the highest values for curvilinear velocity, linear velocity and average velocity. The tested protocol was not effective at maintaining the viability of A. lacustris semen after freezing because no motility was observed in any of the dilutions. However, the Comet Assay demonstrated that cryoprotectant solutions were effective in protecting the genetic material of cells, as DNA damage levels were low, with no difference between control and Me2SO10% + A, dilutions MTG10%+C, Me2SO10%+B and Me2SO15%+B.(AU)

O objetivo deste estudo foi determinar as características do sêmen de Astyanax lacustris após indução hormonal e avaliar a sensibilidade dos espermatozoides da espécie a soluções crioprotetoras baseadas nos crioprotetores dimetilsulfóxido e metilglicol. Volume, cor, concentração espermática, motilidade total e aspectos do movimento espermático foram analisados usando o "Sistema Integrado de Análise de Sêmen (ISAS®CASA)". Três extensores diferentes foram testados: A) glicose 5%+gema de ovo 10%, B) BTS® 5% e C) glicose 5% e dois crioprotetores permeáveis: dimetilsulfóxido (Me2SO) e metilglicol (MTG). O sêmen fresco de A. lacustris apresentou motilidade total 76,6±11,2%, duração da motilidade 33,0±2,2s, concentração de espermatozoides 7,22±3,2×109sptz/mL e osmolalidade seminal 219±0,03mOsm/kg-1. O teste de toxicidade apresentou maiores valores de motilidade total nas diluições MTG15%+A, Me2SO15%+B e Me2SO10%+C, e as diluições Me2SO10%+C e Me2SO15%+C apresentaram maiores valores de velocidade curvilínea, velocidade linear e velocidade média. O protocolo testado não foi eficaz em manter a viabilidade do sêmen de A. lacustris pós-congelamento, pois não foi observada motilidade em nenhuma das diluições. No entanto, o Ensaio Cometa demonstrou que as soluções crioprotetoras eram eficazes na proteção do material genético das células, pois os níveis de dano ao DNA eram baixos, sem diferença entre controle e Me2SO10%+A, MTG10%+C, Me2SO10%+B e Me2SO15%+B.(AU)

Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol

This study aimed to determine the semen characteristics of Astyanax lacustris after hormonal induction and to evaluate the sensitivity of the species sperm to cryoprotective solutions based on the cryoprotectants dimethyl sulfoxide and methyl glycol. Volume, color, sperm concentration, total motility and aspects of sperm movement were analyzed using "Integrated Semen Analysis System". Three different extenders were tested: A) glucose 5%+egg yolk 10%, B) BTS®5% and C) glucose 5% and two permeable cryoprotectants: dimethyl sulfoxide (Me2SO) and methyl glycol (MTG). Fresh A. lacustris semen presented total motility of 76.6±11.2%, motility duration of 33.0±2.2s, sperm concentration of 7.22±3.2×109sptz/mL and seminal osmolality of 219±0.03mOsm/kg-1. The toxicity test showed the highest total motility values at the MTG15%+A, Me2SO15%+B and Me2SO10%+C dilutions, and the Me2SO10%+C and Me2SO15%+C dilutions presented the highest values for curvilinear velocity, linear velocity and average velocity. The tested protocol was not effective at maintaining the viability of A. lacustris semen after freezing because no motility was observed in any of the dilutions. However, the Comet Assay demonstrated that cryoprotectant solutions were effective in protecting the genetic material of cells, as DNA damage levels were low, with no difference between control and Me2SO10% + A, dilutions MTG10%+C, Me2SO10%+B and Me2SO15%+B.(AU)

O objetivo deste estudo foi determinar as características do sêmen de Astyanax lacustris após indução hormonal e avaliar a sensibilidade dos espermatozoides da espécie a soluções crioprotetoras baseadas nos crioprotetores dimetilsulfóxido e metilglicol. Volume, cor, concentração espermática, motilidade total e aspectos do movimento espermático foram analisados usando o "Sistema Integrado de Análise de Sêmen (ISAS®CASA)". Três extensores diferentes foram testados: A) glicose 5%+gema de ovo 10%, B) BTS® 5% e C) glicose 5% e dois crioprotetores permeáveis: dimetilsulfóxido (Me2SO) e metilglicol (MTG). O sêmen fresco de A. lacustris apresentou motilidade total 76,6±11,2%, duração da motilidade 33,0±2,2s, concentração de espermatozoides 7,22±3,2×109sptz/mL e osmolalidade seminal 219±0,03mOsm/kg-1. O teste de toxicidade apresentou maiores valores de motilidade total nas diluições MTG15%+A, Me2SO15%+B e Me2SO10%+C, e as diluições Me2SO10%+C e Me2SO15%+C apresentaram maiores valores de velocidade curvilínea, velocidade linear e velocidade média. O protocolo testado não foi eficaz em manter a viabilidade do sêmen de A. lacustris pós-congelamento, pois não foi observada motilidade em nenhuma das diluições. No entanto, o Ensaio Cometa demonstrou que as soluções crioprotetoras eram eficazes na proteção do material genético das células, pois os níveis de dano ao DNA eram baixos, sem diferença entre controle e Me2SO10%+A, MTG10%+C, Me2SO10%+B e Me2SO15%+B.(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)

Cryopreservation of tambaqui semen using a dry shipper and a programmed freezing machine / Criopreservação do sêmen de tambaqui utilizando dry shipper e máquina de congelação programada

Tambaqui (Colossoma macropomum) is a native freshwater fish that is of great importance for Brazilian aquaculture. Because of this importance, several techniques have been developed to improve the reproduction of this species in captivity. One of these techniques is the cryopreservation of sperm. In an effort to increase the efficiency of cryopreservation protocols, researchers have tried to determine suitable diluting solutions and freezing methods, which will provide a better post-thaw sperm quality. Thus, this study aimed to evaluate the efficiency of different diluents and freezing methods for the cryopreservation of tambaqui (C. macropomum) sperm. Samples of fresh semen were diluted in different treatments (Glucose 5% + 10% Dimethyl sulfoxide DMSO, Glucose 5% + 10% Methyl glycol MG, BTS + 10% DMSO and BTS + 10% MG) at a 1:9 dilution rate and frozen in a programmed freezing machine and a dry shipper. The semen samples were thawed and evaluated for vitality, sperm morphology and kinetics. Cryopreserved semen with DMSO and using the programmed freezing machine provided a greater percentage of motile sperm (15.44 ± 1.04%) after thawing compared to the dry shipper (3.99 ± 0.55%), regardless of the diluent. Additionally, DMSO showed better sperm velocities than MG regardless of the freezing method and the extender employed. A higher percentage of living spermatozoa was obser

O tambaqui (Colossoma macropomum) é uma espécie nativa de peixe de água doce de grande importância para aquicultura brasileira. Devido a isso, diversas técnicas têm sido desenvolvidas para aperfeiçoar a reprodução desta espécie em cativeiro, dentre elas a criopreservação de sêmen de peixe. Como uma forma de melhorar os protocolos de criopreservação, tem-se buscado utilizar soluções diluidoras e métodos de congelação adequados, proporcionando uma boa qualidade seminal pós-descongelação. Dessa forma, este estudo objetivou avaliar a eficiência de diferentes diluidores e métodos de congelação na criopreservação do sêmen de tambaqui (C. macropomum). As amostras de sêmen fresco foram diluídas em diferentes tratamentos (Glicose 5% + 10% Dimetilsufóxido DMSO; Glicose 5% + 10% Metil glicol MG; Beltsville Thawing Solution BTS + 10% DMSO e BTS + 10% MG) na proporção 1:9 e congeladas em máquina de congelação programada e em Dry shipper. As amostras seminais foram descongeladas e avaliadas para vitalidade, morfologia e cinética espermáticas. O sêmen criopreservado com DMSO utilizando a máquina de congelação programada proporcionou maiores percentuais de espermatozoides móveis (15,44 ± 1,04%) após a descongelação em relação ao Dry shipper (3,99 ± 0,55%), independente do diluente utilizado. Além disso, DMSO proporcionou as melhores velocidades espermáticas em relação ao MG, independente

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

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

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

CRIOPRESERVAÇÃO ESPERMÁTICA EM Prochilodus lineatus E Chirostoma estor (Pisces, Prochilontidae e Atherinopsidae)

As construções de barragens, pesca predatória, poluição do habitat e introdução de espécies exóticas são fatores que afetam a reprodução de algumas espécies de peixes e, consequentemente a redução da população selvagem no ambiente. Dessa forma, dois experimentos foram conduzidos para avaliar os efeitos da criopreservação no sêmen de uma espécie modelo animal (Prochilodus lineatus) e de uma espécie ameaçada de extinção (Chirostoma estor). O experimento 1 foi conduzido para avaliar os efeitos da suplementação de diferentes doses de melatonina na criopreservação do sêmen de Prochilodus lineatus. Foram utilizados 18 machos (6 pools de sêmen x 3 machos) e 2 fêmeas de P. lineatus. O meio crioprotetor foi suplementado com 2,00; 2,75; 3,50 e 4,25 mM de melatonina (MEL) e adicionalmente um grupo controle (sem adição de melatonina), totalizando 5 tratamentos e 10 replicatas por tratamentos. Amostras criopreservadas com 2,00 mM MEL exibiram maior taxa de motilidade (93%), em relação aos outros tratamentos com adição de melatonina (6469%). O tratamento controle e 2,00 mM MEL resultaram em maior integridade de membrana (78%) se comparado a 4,25 mM MEL (65%). Em relação ao estresse oxidativo, menor peroxidação lipídica ocorreu nas amostras criopreservadas com 2,75 mM e 3,50 mM MEL (0,72-0,81 M de MDA equivalentes), em comparação com o controle (2,12 M de MDA equivalentes). Enquanto maior atividade enzimática da catalase ocorreu no controle (3,24 U-CAT/mg de proteína). Maiores taxas de fertilização e eclosão ocorreram em 2,75 mM MEL (27% e 17%, respectivamente) em comparação com 4,25 mM MEL (5% e 6%, respectivamente). Por sua vez, no experimento 2, avaliaram-se os efeitos de diluidores e crioprotetores na criopreservação do sêmen de Chirostoma estor. Foram utilizados 42 machos (6 pools de sêmen x 7 machos) e 3 fêmeas de C. estor. Os diluidores comerciais testados foram: BTS, MIII e Androstar Plus e os crioprotetores testados foram: dimetilsulfóxido (DMSO) e metil glicol (MG), constituindo em um experimento fatorial de 3 x 2 (3 diluidores x 2 crioprotetores), totalizando 6 tratamentos e 3 replicatas. Os tratamentos MIII+MG (40%) e Androstar Plus+MG (48%) apresentaram as maiores taxas de motilidade (2030%) que os demais tratamentos. Não foram observadas diferenças quanto a duração de motilidade (114222 s) e integridade de membrana (5460%). Somente para a taxa de fertilização houve interação significativa entre o diluidor e crioprotetor. O crioprotetor MG permitiu taxas de fertilização independente do diluidor (1220%), e apenas em amostras criopreservadas em Androstar Plus+DMSO (14%) foi possível obter taxas de fertilização. Em conclusão, para obter maior qualidade do esperma após o descongelamento, 2,00 mM de melatonina pode ser suplementada ao meio de criopreservação de Prochilodus lineatus, enquanto o sêmen de Chirostoma estor pode ser criopreservado nos diluidores comerciais MIII e Androstar Plus, associado ao crioprotetor metil glicol.

The construction of dams, overfishing, habitat pollution, and the introduction of exotic species are factors that affect the reproduction of some fish species and, consequently, the reduction of the wild population in the environment. Thus, two experiments were carried out to evaluate the effects of cryopreservation in the sperm of an animal model species (Prochilodus lineatus) and an endangered species (Chirostoma estor). Experiment 1 was carried out to evaluate the effects of supplementation of different doses of melatonin on cryopreservation of Prochilodus lineatus sperm. Eighteen males (6 sperm pools x 3 males) and 2 females of P. lineatus were used. The cryoprotective medium was supplemented with 2.00; 2.75; 3.50 and 4.25 mM of melatonin (MEL) and additionally a control group (without the addition of melatonin), totaling 5 treatments and 10 replicates per treatment. Samples cryopreserved with 2.00 mM MEL exhibited a higher motility rate (93%), compared to other treatments with the addition of melatonin (64-69%). The control treatment and 2.00 mM MEL resulted in greater membrane integrity (78%) compared to 4.25 mM MEL (65%). Regarding oxidative stress, lower lipid peroxidation occurred in samples cryopreserved with 2.75 mM and 3.50 mM MEL (0.72-0.81 M MDA equivalents), compared to the control (2.12 M MDA equivalents). While the higher enzymatic activity of catalase occurred in the control (3.24 U-CAT/mg protein). Higher fertilization and hatching rates occurred at 2.75 mM MEL (27% and 17%, respectively) compared to 4.25 mM MEL (5% and 6%, respectively). On the Other hand, in experiment 2, the effects of extenders and cryoprotectants on the sperm cryopreservation of Chirostoma estor were evaluated. Forty-two males (6 semen pools x 7 males) and 3 females of C. estor were used. The commercial extenders tested were: BTS, MIII, and Androstar Plus and the cryoprotectants tested were: dimethylsulfoxide (DMSO) and methyl glycol (MG), constituting a 3 x 2 factorial experiment (3 extenders x 2 cryoprotectants), totaling 6 treatments and 3 replicates. Treatments MIII+MG (40%) and Androstar Plus+MG (48%) had the highest motility rates (20-30%) than the other treatments. No differences were observed in motility duration (114-222 s) and membrane integrity (54-60%). Only for the fertilization rate, there was a significant interaction between extender and cryoprotectant. The MG cryoprotectant allowed dilution rates independent of the extender (12-20%), and only in samples cryopreserved in Androstar Plus+DMSO (14%), it was possible to obtain fertilization rates. In conclusion, to obtain a higher sperm quality after thawing, 2.00 mM of melatonin can be supplemented to the cryopreservation medium of Prochilodus lineatus, while the semen of Chirostoma estor can be cryopreserved in commercial extenders MIII and Androstar Plus associated with cryoprotectant methyl glycol.

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

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

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

Avaliação de diferentes crioprotetores e taxas de diluição na criopreservação seminal de Prochilodus brevis / Evaluation of different crioprotectants and dilution ratios on Prochilodus brevis sperm cryopreservation

Este estudo objetivou caracterizar o sêmen de Prochilodus brevis e avaliar os efeitos de diferentescrioproterores e taxas de diluição sobre a cinética e a morfologia do sêmen criopreservado. Inicialmente,amostras seminais de P. brevis (n = 40) foram analisadas quanto ao volume, pH, osmolaridade, concentração,motilidade e morfologia espermáticas. Para a criopreservação, o material coletado foi distribuído em 10 pools desêmen (n = 10), submetidos a seis tratamentos compostos pela combinação de glicose 5%, dois crioprotetores(DMSO ou MG) e três taxas de diluição (1:3, 1:6 ou 1:9 sêmen:diluidor). As amostras, in natura e pósdescongeladas,foram analisadas quanto à sua morfologia e cinética espermática utilizando o CASA. O sêmen deP. brevis apresentou características semelhantes a demais espécies de Prochilodus. O sêmen congelado comglicose e MG apresentou resultados significativamente superiores (P < 0,05) comparado àquele congeladoutilizando DMSO. As diluições 1:3 e 1:6 apresentaram melhores resultados para glicose + MG, enquanto 1:9 foimelhor para glicose + DMSO. Portanto, a associação glicose + MG, numa taxa de diluição de 1:3 ou 1:6, é amais indicada para a criopreservação do sêmen de Prochilodus brevis.(AU)

This study aimed to characterize Prochilodus brevis sperm and evaluate the effects of different diluentsand dilution ratios on kinetics and morphology of cryopreserved sperm. Initially, sperm samples of P. brevis(n = 40) were assessed for volume, pH, osmolality, spermatic concentration, motility and morphology. Forcryopreservation, the collected material was distributed in ten pools of semen (n = 10), submitted to sixtreatments composed by a combination of glucose 5% with two cryoprotectants (DMSO or MG) and threedilution ratios (1:3, 1:6 or 1:9 - sperm:diluent). In natura and post-thawed samples were assessed for spermaticmorphology and kinetics using CASA. The sperm of P. brevis showed similar characteristics to otherProchilodus species. Sperm cryopreserved in glucose plus MG presented significantly higher results (P < 0.05)compared to sperm frozen in DMSO. The dilution ratios of 1:3 and 1:6 yielded better results for glucose + MG,while 1:9 was the best dilution for glucose + DMSO. In conclusion, the association of glucose + MG, in adilution ratio of 1:3 or 1:6, is the most indicated for Prochilodus brevis sperm cryopreservation.(AU)

Avaliação de diferentes crioprotetores e taxas de diluição na criopreservação seminal de Prochilodus brevis / Evaluation of different crioprotectants and dilution ratios on Prochilodus brevis sperm cryopreservation

Este estudo objetivou caracterizar o sêmen de Prochilodus brevis e avaliar os efeitos de diferentescrioproterores e taxas de diluição sobre a cinética e a morfologia do sêmen criopreservado. Inicialmente,amostras seminais de P. brevis (n = 40) foram analisadas quanto ao volume, pH, osmolaridade, concentração,motilidade e morfologia espermáticas. Para a criopreservação, o material coletado foi distribuído em 10 pools desêmen (n = 10), submetidos a seis tratamentos compostos pela combinação de glicose 5%, dois crioprotetores(DMSO ou MG) e três taxas de diluição (1:3, 1:6 ou 1:9 – sêmen:diluidor). As amostras, in natura e pósdescongeladas,foram analisadas quanto à sua morfologia e cinética espermática utilizando o CASA. O sêmen deP. brevis apresentou características semelhantes a demais espécies de Prochilodus. O sêmen congelado comglicose e MG apresentou resultados significativamente superiores (P < 0,05) comparado àquele congeladoutilizando DMSO. As diluições 1:3 e 1:6 apresentaram melhores resultados para glicose + MG, enquanto 1:9 foimelhor para glicose + DMSO. Portanto, a associação glicose + MG, numa taxa de diluição de 1:3 ou 1:6, é amais indicada para a criopreservação do sêmen de Prochilodus brevis.

This study aimed to characterize Prochilodus brevis sperm and evaluate the effects of different diluentsand dilution ratios on kinetics and morphology of cryopreserved sperm. Initially, sperm samples of P. brevis(n = 40) were assessed for volume, pH, osmolality, spermatic concentration, motility and morphology. Forcryopreservation, the collected material was distributed in ten pools of semen (n = 10), submitted to sixtreatments composed by a combination of glucose 5% with two cryoprotectants (DMSO or MG) and threedilution ratios (1:3, 1:6 or 1:9 - sperm:diluent). In natura and post-thawed samples were assessed for spermaticmorphology and kinetics using CASA. The sperm of P. brevis showed similar characteristics to otherProchilodus species. Sperm cryopreserved in glucose plus MG presented significantly higher results (P < 0.05)compared to sperm frozen in DMSO. The dilution ratios of 1:3 and 1:6 yielded better results for glucose + MG,while 1:9 was the best dilution for glucose + DMSO. In conclusion, the association of glucose + MG, in adilution ratio of 1:3 or 1:6, is the most indicated for Prochilodus brevis sperm cryopreservation.

SPERM CRYOPRESERVATION THROUGHOUT THE SPAWNING SEASON IN Prochilodus lineatus

O objetivo deste trabalho foi determinar a qualidade do sêmen de curimba (Prochilodus lineatus) criopreservado ao longo do período reprodutivo (novembro a março). O sêmen foi coletado mensalmente dos animais (43 machos) após indução hormonal com extrato bruto de hipófise de carpa. O sêmen fresco foi avaliado subjetivamente quanto a sua motilidade, vigor, duração da motilidade e morfologia. O sêmen foi criopreservado em palhetas de 0,5 mL (n=258) utilizando-se metilglicol como crioprotetor e solução de glicose como diluidor. O sêmen descongelado foi avaliado quanto a sua motilidade, velocidades (curvilínea VCL, linear progressiva VSL, média VAP) e frequência de batimento flagelar cruzado (BCF) por meio de um sistema de análise espermática computadorizada (CASA). O sêmen descongelado também foi analisado quanto a sua morfologia, estresse oxidativo (peroxidação lipídica LPO, espécies reativas de oxigênio ROS, superóxido desmutase SOD e catalase CAT) e capacidade de fertilização (taxas de fertilização e eclosão). O plasma seminal foi analisado quanto ao seu pH, osmolalidade e concentração iônica (Na+, K+, Ca2+ e Mg2+). O sêmen fresco de curimba apresentou alta qualidade durante o período reprodutivo, com taxa de motilidade espermática superior a 80%, e a qualidade do sêmen congelado foi afetada ao longo da estação reprodutiva. O sêmen criopreservado apresentou maiores (p<0.05) motilidade e VCL de dezembro a março. Correlações negativas (p<0.01) foram observadas entre a concentração espermática e a motilidade espermática, concentração espermática e a VCL, e concentração de íons Ca2+ no plasma seminal e motilidade espermática. A concentração espermática foi positivamente correlacionada (p<0.01) com a concentração de íons Ca2+ no plasma seminal, CAT e ROS. CAT e fertilidade também se correlacionaram positivamente (p<0.05). O aumento da CAT foi eficiente em reduzir as ROS e a LPO nas amostras criopreservadas em novembro, e manteve a capacidade de fertilização espermática, apesar de a atividade das ROS ter afetado a motilidade e a VCL. Fatores climáticos registrados no período que antecedeu a estação reprodutiva também afetaram a qualidade das amostras criopreservadas no início do período. Apesar de o sêmen fresco e criopreservado terem apresentado baixo percentual de células normais em março, as amostras apresentaram boa qualidade neste mês, com alta motilidade, vigor e VCL. O sêmen de P. lineatus criopreservado de dezembro a março exibe características mais favoráveis para ser submetido ao estresse induzido pela criopreservação.

The aim of this study was to determine the quality of Prochilodus lineatus sperm cryopreserved throughout the reproductive season (November to March). Males (n=43) were monthly handstripped after carp pituitary treatment. Fresh sperm was subjectively analyzed for its motility rate, motility quality score, duration of motility and morphology. Sperm was cryopreserved on 0.5 mL straws (n=258) using methyl glycol as cryoprotectant and glucose solution as extender. Post-thaw sperm was analyzed for its motility rate, velocities (curvilinear VCL; straight-line VSL; average path VAP), and beat cross frequency (BCF) using a Computer-Assisted Sperm Analyzer (CASA). Frozen sperm morphology, oxidative stress (lipid peroxidation LPO; reactive oxygen species ROS; superoxide dismutase SOD; catalase CAT), and fertilization capacity (fertilization and hatching rates) were also evaluated. Seminal plasma was analyzed for pH, osmolality and ion concentration (Na+, K+, Ca2+ and Mg2+). P. lineatus fresh sperm presented good quality during the reproductive season, exhibiting motility rates above 80%, and post-thaw sperm quality was affected throughout the spawning season. Post-thaw sperm yielded higher (p<0.05) motility and VCL on December to March. Negative correlations were observed between sperm concentration and frozen sperm motility, sperm concentration and VCL, and Ca2+ concentration on seminal plasma and frozen sperm motility (p<0.01). Sperm concentration was positively correlated with Ca2+ concentration, CAT and ROS (p<0.01). CAT and fertility correlated positively (p<0.05). Increased CAT was efficient in reducing ROS and LPO in samples frozen in November, and maintained sperm fertilization capacity, although ROS activity affected sperm motility and VCL. Climate factors registered before the reproductive season also affected the quality of samples frozen at the beginning of the season. Although both fresh and post-thaw semen presented lower percentages of normal sperm cells on March, samples yielded good quality parameters such as motility rate, motility quality score, and VCL on this month. P. lineatus sperm cryopreserved from December to March exhibits better characteristics to undergo the stress induced by cryopreservation.

Testis evaluation of adult Wistar rats after neonatal treatment with fluoxetine / Avaliação testicular de ratos Wistar adultos após tratamento neonatal com fluoxetina

In current assay the serotoninergic system in newly-born Wistar rats underwent pharmacological modification by fluoxetine, a selective serotonin reuptake inhibitor (SSRI), to investigate its repercussion on testicular parameters in adult animals. Thirty animals were distributed according to treatment: control animals (n = 6), animals treated with 1 mg kg-1 (n = 6), 5 mg kg-1 (n = 6), 10 mg kg-1 (n = 6) and 20 mg kg-1 (n = 6) of fluoxetine (IP). When 150 days old, the animals were anesthetized and perfused intra-cardiacally with fixative solution. Testes were routinely processed for inclusion in plastic resin (methacrylate glycol). Further, 4 m-thick histological sections were stained with toluidine blue/sodium borate 1% and analyzed histometrically. Pharmacological intervention on the serotoninergic system during the postnatal period of the testes development in Wistar rats with fluoxetine chlorohydrate reduced parameters, such as testicular weight, testis liquid weight and seminiferous tubules diameter. However, testicular parameters, such as daily sperm production (DSP), spermatogenesis efficiency (DSP/g/testis) and cell population in stage VII of adult animals, were not influenced by fluoxetine chlorohydrate usage during neonatal period. Results show that administration of fluoxetine during 21 days after birth may induce adverse changes in the spermatogenesis of adult rats.(AU)

No presente trabalho, o sistema serotoninérgico de ratos Wistar recém-nascidos foi farmacologicamente modificado por um inibidor seletivo de recaptação da serotonina, fluoxetina, com o objetivo de observar sua repercussão nos parâmetros testiculares em ratos adultos. Trinta animais foram distribuídos de acordo com o tratamento: controle (n = 6), tratado com 1 mg kg-1 (n = 6), 5 mg kg-1 (n = 6), 10 mg kg-1 (n = 6) e 20 mg kg-1 (n = 6) de fluoxetina. Aos 150 dias de vida, os animais foram anestesiados e perfundidos intracardiacamente com solução fixadora. Os testículos foram removidos e processados para inclusão em resina plástica (glicol metacrilato). Cortes histológicos com 4 m de espessura foram corados por azul de toluidina/borato de sódio 1% e analisados histometricamente. O tratamento com fluoxetina reduziu nos parâmetros de peso testicular líquido e bruto, bem como no diâmetro dos túbulos seminíferos. Entretanto, os parâmetros testiculares de produção espermática diária (PED), eficiência espermática (PED/g/testículo) e população de células germinativas no estágio VII não estavam alteradas pelo tratamento com fluoxetina. Em conclusão, a administração de fluoxetina durante 21 dias após o nascimento pode induzir efeitos adversos na espermatogênese de ratos adultos.(AU)

Osmolalidade das soluções ativadoras com ou sem íons na ativação da motilidade do sêmen criopreservado de piabanha Brycon insignis (Characiformes)

O objetivo deste estudo foi determinar o efeito de soluções ativadoras com diferentes osmolalidades, com ou sem íons, na qualidade espermática do sêmen criopreservado de Brycon insignis. O sêmen de 8 machos foi coletado após tratamento hormonal com extrato de hipófise de carpa. Após a coleta, o sêmen foi criopreservado utilizando metilglicol como crioprotetor e uma solução de BTS® como diluidor. O sêmen diluído foi envasado em palhetas de 0,5 mL, que foram congeladas em botijão de vapor de nitrogênio (dryshipper) e armazenadas em botijão de nitrogênio líquido. Após cinco anos, as palhetas foram descongeladas em banho-maria a 60°C por 8 segundos. No experimento 1, 11 soluções foram preparadas utilizando água osmose reversa (~ 0 mOsm/Kg) e glicose ou NaCl ajustadas as seguintes osmolalidades: 50, 100, 150, 200 e 250 mOsm/Kg. No experimento 2, 6 soluções foram preparadas utilizando água osmose reversa e NaHCO3, citrato de sódio (Na3C6H5O7), NaCl, KCl, CaCl2 ou glicose (controle não iônico), ajutadas a ~ 98 mOsm/Kg. Os parâmetros de taxa de motilidade, velocidade curvilínea (VCL), velocidade retilínea (VSL), velocidade média de percurso (VAP) e frequência de batimento flagelar cruzado (BCF) do sêmen após o descongelamento foram determinados utilizando o sistema computadorizado de análise de sêmen (CASA). Maiores taxas de motilidade e velocidades foram observadas em amostras ativadas nas soluções de NaCl e glicose em osmolalidades de 0 a 200 mOsm/Kg, se comparadas com as amostras ativadas nas soluções de 250 mOsm/Kg e não foram observadas diferenças estatísticas entre as soluções de NaCl e glicose dentro da mesma osmolalidade. As amostras ativadas em NaHCO3, citrato de sódio, NaCl, KCl e glicose apresentaram as maiores taxas de motilidade, se comparada às amostras ativadas em CaCl2. Maiores velocidades curvilineares foram observadas em amostras ativadas em NaHCO3, citrato de sódio, KCl e glicose, quando comparadas às amostras ativadas em CaCl2. As amostras ativadas em NaCl apresentaram valores intermediários para VCL. O sêmen criopreservado de B. insignis pode ser ativado em soluções iônicas ou não iônicas com osmolalidades de 0 a 200 mOsm/Kg, entretanto a solução de CaCl2 deve ser evitada.

The objective of this study was to determine the effect of activating solutions with different osmolalities, with or without ions, on the sperm quality of the cryopreserved sperm of Brycon insignis. The semen of 8 males was collected after hormonal treatment with carp pituitary extract. After collection, the semen was cryopreserved using methyl glycol as a cryoprotectant and a BTS solution was used as extender. The diluted sperm loaded into 0.5 mL straws, which were frozen in a nitrogen vapor vessel (dry shipper) and stored in a liquid nitrogen vessel. After five years the straws were thawed in a water bath at 60 °C for 8 seconds. In experiment 1, 11 solutions were prepared with reverse osmosis water (0 mOsm kg-1) and glucose or NaCl adjusted to an osmolality of 50, 100, 150, 200 and 250 mOsm kg-1. In experiment 2, 6 solutions were prepared with reverse osmosis water e NaHCO3, sodium citrate ((Na3C6H5O7), NaCl, KCl, CaCl2 or glicose (as an ion-free control), adjusted to ~98 mOsm Kg-1. The parameters of motility rate, curvilinear velocity (VCL), straight line (VSL), average path (VAP) and beat-cross frequency (BCF) of the post-thaw were determined using a Computer-Assisted Sperm Analyzer (CASA). Higher motility rates and velocities were observed in activated samples in NaCl and glucose solutions at osmolalities of 0 to 200 mOsm Kg-1, when compared to the activated samples in the solutions of 250 mOsm Kg-1 and was no statistical difference on motility rate and velocities between samples activated in NaCl or in glucose solutions within the same osmolality. The samples activated in NaHCO3, sodium citrate, NaCl, KCl or glucose presented the higher rates of motility, when compared to samples activated in CaCl2. Higher curvilinear velocities were observed in samples activated in NaHCO3, sodium citrate, KCl and glucose, when compared to samples activated in CaCl2. Samples activated in NaCl presented intermediate VCL values. The post-thaw sperm in B. insignis can be activated in ionic or non-ionic solutions with osmolalities of 0 to 200 mOsm/kg, however the CaCl2 solution should be avoided.

Viability and fertility of stallion semen frozen with ethylene glycol and acetamide as a cryogenic agent

This study was designed to determine the effect of cooling at a rate of - 0.13 o C/min or any slow cooling prior to freezing on stallion sperm and to determine th e effect of two thawing temperatures on the viability of sperm frozen using a lactose - EDTA - egg yolk extender (LAC) with 3.5% ethylene glycol (LAC 3.5% EG) or a LAC with the additional incorporation of methyl cellulose, trehalose and acetamide (LAC+5% AC). No differences were observed in the post - thaw parameters of sperm frozen using either one of the cryogenic agents or frozen with or without previous slow cooling. Thawing at 75 o C was better than at 37 o C (P < 0.05). An interaction was observed between the c ryoprotectant and the freezing protocol (P < 0.05). A conception rate of 23.3% was obtained after AI using equine semen frozen with LAC+5% AC.

Viability and fertility of stallion semen frozen with ethylene glycol and acetamide as a cryogenic agent

This study was designed to determine the effect of cooling at a rate of - 0.13 o C/min or any slow cooling prior to freezing on stallion sperm and to determine th e effect of two thawing temperatures on the viability of sperm frozen using a lactose - EDTA - egg yolk extender (LAC) with 3.5% ethylene glycol (LAC 3.5% EG) or a LAC with the additional incorporation of methyl cellulose, trehalose and acetamide (LAC+5% AC). No differences were observed in the post - thaw parameters of sperm frozen using either one of the cryogenic agents or frozen with or without previous slow cooling. Thawing at 75 o C was better than at 37 o C (P < 0.05). An interaction was observed between the c ryoprotectant and the freezing protocol (P < 0.05). A conception rate of 23.3% was obtained after AI using equine semen frozen with LAC+5% AC.(AU)

Meios de congelação para conservação de sêmen de peixes da família characidae / Cryopreservation extenders for fishes sperm of Characidae family

Os peixes caracídeos possuem grande diversidade de espécies, considerável importância ecológica e expressivo valor comercial. A criopreservação de sêmen é uma técnica utilizada para contribuir na operacionalização dos programas de reprodução dessas espécies cultivadas ou em vias de extinção. Para garantir a sobrevivências dos espermatozóides durante a criopreservação é necessário adicionar soluções diluidoras e crioprotetoras, que reagem com o sêmen de forma peculiar dependendo da espécie animal. Portanto, o objetivo dessa revisão foi buscar identificar diluentes adotados para criopreservação de sêmen de alguns peixes caracídeos. Os diluidores mais utilizados são glicose, BTS e, mais recentemente, a água de coco em pó (ACP®) associados aos crioprotetores DMSO, glicerol ou metilglicol. Diante da grande variedade de protocolos, conclui-se que é de grande importância incentivar o estudo das particularidades do sêmen de caracídeos e dos meios de congelação, para padronizar a técnica de criopreservação de sêmen de peixes deste grupo.

family has a great diversity of species, an enormous ecological importance and commercial value. The sperm cryopreservation is a technique used to assist reproductive programs of cultivated or endangered species. To ensure the sperm survival during cryopreservation, it is necessary to add extenders and cryoprotective solutions, which react with the sperm in a peculiar way, depending on the animal specie. Thus, the objective of this review was to identify extenders used in the cryopreservation technique for Characid fishes. The sperm cryopreservation review in Characid fishes report that the most commonly used extenders are glucose, BTS and powder coconut water (PCW®) associated with glycerol, DMSO and methyl glycol. Due to the wide range of protocols, it can be concluded that is very important to encourage the study of the particularities of Characid fish semen and freezing substances to standardization of cryopreservation techniques of semen in this particular fish group.

Extender composition, osmolality, cryoprotectan t and equilibration time effects on fresh sperm motility of two Characiformes fish: piracanjuba ( Brycon orbignyanus ) and streaked prochilod ( Prochilodus lineatus )

Studies regarding the effects of extender composition, osmolality, cryoprotectant (CPA) and equilibration time on the induction/suppression of sperm motility are necessary to establish standard activating agents and immobilizing media for improving both artificial fertilization and pr eservation techniques. Thus, the aim of this study was to evaluate the effects of these factors on fresh sperm motility in piracanjuba ( Brycon orbignyanus ) and streaked prochilod ( Prochilodus lineatus ). Twenty four media, as a combination of six extenders (BTS™ and glucose solutions at 270, 315 and 360 mOsm/kg) with the CPAs DMSO, methanol, methyl glycol (MG) and a control without CPA, were prepared. Immediately after dilution, samples were observed under a light microscope to confirm whether different extender-CPA combinations would suppress the initiation of sperm motility. Motility was then triggered in 92 mOsm/kg NaCl and evaluated immediately after dilution (non-equilibrated samples) and after a 30-min equilibration time at 4°C for motility rate and motility quality score (0 = no movement; 5 = rapidly swimming sperm). In both species, motility was initiated in all samples diluted in BTS-270-control, Glu-270-MG, Glu- 270-control and in all combinations containing DMSO. In B. orbignyanus , motility rate (77 to 92%) and motility quality score (3.3 to 4.7) of non-equilitrated samples was not significantly affected by any parameter. After 30 min, however, motility quality score decreased in most of the samples, mainly when diluted in BTS™ (3.3 to 4.2). In P. lineatus , motility rate was significantly higher in non-equilibrated samples (overall mean = 83%) compared to 30-min equilibrated samples (overall mean = 75%). Motility quality score of non- equilibrated samples was not affected by any parameter (3.3 to 4.2), but samples equilibrated in DMSO yielded the lowest score (3.0). Sperm motility (rate and score) was affected differently in B. orbignyanus compared to P. lineatus , and this finding should be considered when developing a methodology for sperm cryopreservation.

Meios de congelação para conservação de sêmen de peixes da família characidae / Cryopreservation extenders for fishes sperm of Characidae family

Os peixes caracídeos possuem grande diversidade de espécies, considerável importância ecológica e expressivo valor comercial. A criopreservação de sêmen é uma técnica utilizada para contribuir na operacionalização dos programas de reprodução dessas espécies cultivadas ou em vias de extinção. Para garantir a sobrevivências dos espermatozóides durante a criopreservação é necessário adicionar soluções diluidoras e crioprotetoras, que reagem com o sêmen de forma peculiar dependendo da espécie animal. Portanto, o objetivo dessa revisão foi buscar identificar diluentes adotados para criopreservação de sêmen de alguns peixes caracídeos. Os diluidores mais utilizados são glicose, BTS e, mais recentemente, a água de coco em pó (ACP®) associados aos crioprotetores DMSO, glicerol ou metilglicol. Diante da grande variedade de protocolos, conclui-se que é de grande importância incentivar o estudo das particularidades do sêmen de caracídeos e dos meios de congelação, para padronizar a técnica de criopreservação de sêmen de peixes deste grupo.(AU)

family has a great diversity of species, an enormous ecological importance and commercial value. The sperm cryopreservation is a technique used to assist reproductive programs of cultivated or endangered species. To ensure the sperm survival during cryopreservation, it is necessary to add extenders and cryoprotective solutions, which react with the sperm in a peculiar way, depending on the animal specie. Thus, the objective of this review was to identify extenders used in the cryopreservation technique for Characid fishes. The sperm cryopreservation review in Characid fishes report that the most commonly used extenders are glucose, BTS and powder coconut water (PCW®) associated with glycerol, DMSO and methyl glycol. Due to the wide range of protocols, it can be concluded that is very important to encourage the study of the particularities of Characid fish semen and freezing substances to standardization of cryopreservation techniques of semen in this particular fish group.(AU)

Extender composition, osmolality, cryoprotectan t and equilibration time effects on fresh sperm motility of two Characiformes fish: piracanjuba ( Brycon orbignyanus ) and streaked prochilod ( Prochilodus lineatus )

Studies regarding the effects of extender composition, osmolality, cryoprotectant (CPA) and equilibration time on the induction/suppression of sperm motility are necessary to establish standard activating agents and immobilizing media for improving both artificial fertilization and pr eservation techniques. Thus, the aim of this study was to evaluate the effects of these factors on fresh sperm motility in piracanjuba ( Brycon orbignyanus ) and streaked prochilod ( Prochilodus lineatus ). Twenty four media, as a combination of six extenders (BTS™ and glucose solutions at 270, 315 and 360 mOsm/kg) with the CPAs DMSO, methanol, methyl glycol (MG) and a control without CPA, were prepared. Immediately after dilution, samples were observed under a light microscope to confirm whether different extender-CPA combinations would suppress the initiation of sperm motility. Motility was then triggered in 92 mOsm/kg NaCl and evaluated immediately after dilution (non-equilibrated samples) and after a 30-min equilibration time at 4°C for motility rate and motility quality score (0 = no movement; 5 = rapidly swimming sperm). In both species, motility was initiated in all samples diluted in BTS-270-control, Glu-270-MG, Glu- 270-control and in all combinations containing DMSO. In B. orbignyanus , motility rate (77 to 92%) and motility quality score (3.3 to 4.7) of non-equilitrated samples was not significantly affected by any parameter. After 30 min, however, motility quality score decreased in most of the samples, mainly when diluted in BTS™ (3.3 to 4.2). In P. lineatus , motility rate was significantly higher in non-equilibrated samples (overall mean = 83%) compared to 30-min equilibrated samples (overall mean = 75%). Motility quality score of non- equilibrated samples was not affected by any parameter (3.3 to 4.2), but samples equilibrated in DMSO yielded the lowest score (3.0). Sperm motility (rate and score) was affected differently in B. orbignyanus compared to P. lineatus , and this finding should be considered when developing a methodology for sperm cryopreservation.(AU)

Comparative pathologic and stereologic study of respiratory system in mice following administration of 3-methyl indole dissolved in propylene glycol and cremophor EL and LD50 determination

Background: 3-Methyl indole (Skatole, 3-MI) is produced by microfloral fermentation of tryptophan in rumen and production of that has relation with the onset of respiratory problems in cattle. The aim of this study was to compare complications of 3-MI dissolved in two solvents, Cremophor and Propylene Glycol on BALB/c mice by histopathologic and stereologic studies. Materials, Methods & Results: Female Balb/C mice 56 days of age (23 - 27 g) were divided into 13 groups of eight and had access to food and water ad libitum. Mice were housed in plastic cages (4 per cage) with wire bar lids on bedding of sawdust in an air-conditioned room with 12 h light: dark cycles. After a 7-day acclimation period, for 5 groups, Mice were injected with 400, 500, 600, 700 and 800 mg/kg from solution of 3-MI in propylene glycol and similarly for 5 other groups with Cremophor. As well as 2 groups were injected with Propylene glycol and Cremophor with the dosage that is necessary for solving the highest dose of 3-MI. One group received normal saline with the volume equal with Cremophor (drug vehicle) group. Briefly, 125 mg of 3-MI was dissolved per mL of sterile filtered Propylene glycol and 30 mg of 3-MI was dissolved per mL of sterile filtered Cremophor. Once injected intra peritoneally, mice were monitored 3-4 h for external reaction signs from the injection. LD50 was determined 610.16 mg/kg 3-Methylindole and 687.5 mg/kg 3-Methylindole dissolved in Propylene glycol and Cremophor respectively with linear regression. Increase in respiration rate, tachypnea and shallow respiration was observed in clinical examination. In necropsy, alveolar and bronchial hyperemia was significant in lungs. Some superficial wounds were observed in upper respiratory airways. Trachea and Hungarian airways were severely hyperemic. There were some debriss in airways canals. We couldnt find any lesion in other organs. Histopathology showed noticeable alveolar edema and emphysema, type I pneumocytes necrosis, Slight endothelial lesions, rare degeneration of type II pneumocytes and raised nutrophil infiltration around vessels were observed in higher dosages of both groups. Moderate interstitial edema, hyperemia and increase of alveolar macrophages were observed too. Increase of lymphocytes was noticed after the dose 600 mg/kg moderately on a dose dependent manner. There was a Hyaline membrane in the wall of air sacs. Endothelial capillary damage in Cremophor dissolved 3-MI groups were slightly lower. Although damage to the vessels was not that much progressed in both groups. Stereologic study of type II pneumocytes showed a significant increase in the percentage of them in alveoli of treatment groups in a dose dependent manner compare to control and drug vehicle groups. At the highest dose (800 mg/kg) the group with Propylene glycol as solvent had significant difference with Cremophor dissolved 3-Methyl indole group and epithelialization was more severe in that. Bronchiole injuries raised in a dose dependent manner in both treatment groups. Discussion: The results of this research indicate that intraperitoneal infusion of 3-MI dissolved in Propylene glycol and Cremophor cause noticeable changes in the respiratory systems of mice. Effects of 3-MI in Propylene glycol is more severe than 3-MI dissolved in Cremophor but type of the lesions is not different. Bronchiolar lesions were moderate compare to the effects of 3-MI in some other animals. Severity of vascular lesions in Propylene glycol dissolved 3-MI was greater than groups that received the Cremophor dissolved 3-MI. Although the vascular injuries were not that much noticeable at all.(AU)