Efeito de diferentes osmolaridades do diluidor Tris-citrato na congelação com etilenoglicol ou glicerol de espermatozoides epididimários de gato doméstico (Felis catus) / Effect of different osmolarities of Tris-citrate extender on freezing epididymal sperm from domestic cat (Felis catus)with ethyleneglycol or glycerol

Objetivou-se com o estudo investigar o efeito de diferentes osmolaridades do diluidor Triscitrato em espermatozóides epididimários de gatos domésticos (Feliscatus) e a congelação com glicerol ou etilenoglicol. Foram realizados dois experimentos, com 10 gatos. No Experimento 1, avaliou-se a manutenção dos parâmetros espermáticos em diluidor tris-citrato com osmolaridades 275, 325, 375, 425, 475 e 525mOsm, nos tempos (T0= 0, T1= 30 e T2= 60 min). No Experimento 2 a congelação foi realizada utilizando as osmolaridades 325 e 375 do glicerol a 4% ou etilenoglicol a 3%, 6%. Dentre as osmolaridades, quanto à motilidade a 325 mOsm não diferiu estatisticamente com o fluido epidídimal (controle) nos três tempos e a 375 mOsm no T0 e T1, e ambas não apresentaram diferenças estatísticas entre si e entre os tempos em todos os parâmetros espermáticos. O uso de 4% de glicerol em diluidor com 375 mOsm foi superior, apresentando motilidade de 25% ± 6, vigor 4, integridade de membrana plasmática de 48% ± 9, sem diferenças estatísticas com o resfriamento e na morfologia não foram encontradas diferenças estatísticas entre as duas osmolaridades. Portanto, o Tris-citrato com 325 e 375 mOsm entre as osmolaridades testadas e pós congelação com 375 mOsm e glicerol 4% manteve os parâmetros espermáticos.(AU)

The aim of this study was to investigate the effect of different osmotic potentials of Tris-citrate extender in epididymal spermatozoa of domestic cats (Felis catus), frozen with glycerol or ethyleneglycol. Two experiments were carried out, with ten cats. In the first experiment, the influence of extender with the osmolarityof 275, 325, 375, 425, 475 and 525 mOsm on sperm parameters were evaluated. In the second experiment, slow freezing was performed using glycerol at 4% or ethyleneglycolat 3% and 6% added to extender with 325and 375 mOsm. Among the osmolarities, the motilityat 325 mOsm did not differ statistically with epididymal fluid (control) at all times evaluated and at 375 mOsmat T0 and T1, and both showed no statistical differences between each other and between the times in all sperm parameters. Glycerol 4% added to extender with 475 mOsm was superior, presenting motilityof 25% ± 6, vigor 4, plasma membrane integrity of 48% ± 9, without statistical differences with cooling and in morphology, no statistical differences were found between the two osmolarities. Therefore, 325 and 375 mOsm Triscitrate between the osmolarities tested and after freezing with 375 mOsm and 4%, glycerol maintained the sperm parameters.(AU)

Efeito de diferentes osmolaridades do diluidor Tris-citrato na congelação com etilenoglicol ou glicerol de espermatozoides epididimários de gato doméstico (Felis catus) / Effect of different osmolarities of Tris-citrate extender on freezing epididymal sperm from domestic cat (Felis catus)with ethyleneglycol or glycerol

Objetivou-se com o estudo investigar o efeito de diferentes osmolaridades do diluidor Triscitrato em espermatozóides epididimários de gatos domésticos (Feliscatus) e a congelação com glicerol ou etilenoglicol. Foram realizados dois experimentos, com 10 gatos. No Experimento 1, avaliou-se a manutenção dos parâmetros espermáticos em diluidor tris-citrato com osmolaridades 275, 325, 375, 425, 475 e 525mOsm, nos tempos (T0= 0, T1= 30 e T2= 60 min). No Experimento 2 a congelação foi realizada utilizando as osmolaridades 325 e 375 do glicerol a 4% ou etilenoglicol a 3%, 6%. Dentre as osmolaridades, quanto à motilidade a 325 mOsm não diferiu estatisticamente com o fluido epidídimal (controle) nos três tempos e a 375 mOsm no T0 e T1, e ambas não apresentaram diferenças estatísticas entre si e entre os tempos em todos os parâmetros espermáticos. O uso de 4% de glicerol em diluidor com 375 mOsm foi superior, apresentando motilidade de 25% ± 6, vigor 4, integridade de membrana plasmática de 48% ± 9, sem diferenças estatísticas com o resfriamento e na morfologia não foram encontradas diferenças estatísticas entre as duas osmolaridades. Portanto, o Tris-citrato com 325 e 375 mOsm entre as osmolaridades testadas e pós congelação com 375 mOsm e glicerol 4% manteve os parâmetros espermáticos.

The aim of this study was to investigate the effect of different osmotic potentials of Tris-citrate extender in epididymal spermatozoa of domestic cats (Felis catus), frozen with glycerol or ethyleneglycol. Two experiments were carried out, with ten cats. In the first experiment, the influence of extender with the osmolarityof 275, 325, 375, 425, 475 and 525 mOsm on sperm parameters were evaluated. In the second experiment, slow freezing was performed using glycerol at 4% or ethyleneglycolat 3% and 6% added to extender with 325and 375 mOsm. Among the osmolarities, the motilityat 325 mOsm did not differ statistically with epididymal fluid (control) at all times evaluated and at 375 mOsmat T0 and T1, and both showed no statistical differences between each other and between the times in all sperm parameters. Glycerol 4% added to extender with 475 mOsm was superior, presenting motilityof 25% ± 6, vigor 4, plasma membrane integrity of 48% ± 9, without statistical differences with cooling and in morphology, no statistical differences were found between the two osmolarities. Therefore, 325 and 375 mOsm Triscitrate between the osmolarities tested and after freezing with 375 mOsm and 4%, glycerol maintained the sperm parameters.

Influence of Cooling Time and Diluents on the Freezability of Prochilodus brevis semen

Background: Prochilodus brevis is a rheophilic fish of economic and ecological importance. However, anthropic action has made its population vulnerable. Thus, the development of reproductive biotechnologies, such as seminal conservation, is necessary to subsidize their fish farming. However, seminal collections are often performed in places with few laboratory resources, demanding studies to determine the maximum time for which sperm can be cooled, as well as its process until frozen. Thus, the present study aimed to evaluate the influence of cooling time and the presence of dilution solutions on cryopreservation of P. brevis semen.Material, Methods & Results: After seminal collection, nine pools were formed and analyzed for seminal pH, concentration, membrane integrity, morphology and spermatic kinetics - motility, curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). After the analysis of the pools in natura (control 1), they were processed as follows: 1)- immediate freezing (control 2); 2)- cooling: undiluted, diluted in coconut water powder (ACP-104) or diluted in 5% glucose, followed by cooling at different times (6, 12, 24 or 48 h); 3)- Post-refrigeration freezing: the pools were diluted in their respective diluents and 10% dimethyl sulfoxide. After 15 days, the samples were thawed and analyzed for the aforementioned parameters. For

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

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

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

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

Efeito da melatonina sobre a integridade das membranas plasmática e mitocondrial de espermatozoides criopreservados de ovino / Effect of Melatonin on the plasmatic and mitochondrial integrity membrane of sperm cryopreserved ram

This study evaluate the effect of melatonin supplementation to TRIS-GEMA diluter in plasmatic andmitochondria membrane integrity of semen ram cryopreserved. Was used seven ejaculates of six ram,whichwere pooled in pool, according to the experimental groups: Control; T1 - 1x10-7 mol / L and T2 - 1x10-9 mol / Lmelatonin. Posteriorly the diluting the samples were packaged in straws of 0.25 ml and cryopreserved. Afterthawing at 37°C for 30 seconds, the samples were analyzed for Test thermoresistance slow (TTR) andacrosomal membrane integrity. For statistical analysis we used the Duncan test (α = 0.05) for meancomparison. Treatments 1 and 2 differed statistically control for plasma membrane integrity at times 60, 120and 180min. It was concluded that supplementation 1x10-7mol / L and 1x10-9mol / L of melatonin increased theintegrity of the plasma membrane to 60,120 and 180 minutes.(AU)

Efeito da melatonina sobre a viabilidade de espermatozóides criopreservados de ovinos / Effect of melatonin on the feasibility of sperm cryopreserved of ram

This study to evaluate the effect of melatonin supplementation to TRIS-GEMA diluter in sperm viabilityof semen ram cryopreserved. Was used seven ejaculates of six ram,which were pooled in pool, according to theexperimental groups: T1 - control; T2 - 1x10-7 mol / L and T3 - 1x10-9 mol / L melatonin. Posteriorly thediluting the samples were packaged in straws of 0.25 ml and cryopreserved. After thawing at 37°C for 30seconds, the samples were analyzed for Test thermoresistance slow (TTR) and acrosomal membrane integrity.For statistical analysis we used the Duncan test (α = 0.05) for mean comparison. The results demonstrated thatto the TTR at time 0 min, T2 showed 54.28% motility, and T1obtained lower values in the times 0 and 60 minutesfor force. Supplementation of 1x10-9mol / L of melatonin improved total motility.(AU)

Avaliação da integridade da membrana plasmática e do potencial de atividade mitocondrial de sêmen criopreservado de touros frente a ação do (Limoneno (R)-(+) / Evaluation of plasma membrane integrity and mitochondrial activity potential of cryopreserved semen of bulls on action of the Limonene (R) - (+)

This study was conducted to evaluate the effect of different concentrations of limonene (R)-(+) (50 µM,100 µM and 150 µM) in supplementing the diluter of bulls freezing semen. Thirty-six ejaculated from fourCurraleiro-Pé-Duro bulls were used for cryopreservation. The epifluorescence microscopy was used todetermine the plasmatic integrity and mitochondrial activity potential. No was observed effect on the integrity ofplasma membrane nor mitochondrial activity potential when cryopreserved by adding limonene R - (+). Theresults obtained in this study allow to conclude that supplementation limonene (R) - (+) on diluter of bullsfreezing semen does not interfere with the integrity of the plasma membrane or the potential of spermmitochondrial activity.(AU)

Ação do Limoneno (S)-(-) sobre a cinética espermática de sêmen criopreservado de touros / Action of the Limonene (S) - (-) on sperm kinetics of cryopreserved semen of bulls

This study was conducted to evaluate the effect of different concentrations of Limoneno (S)-(-) (50 µM,100 µM and 150 µM) in supplementing the diluter of bulls freezing semen. Thirty-six ejaculated from fourCurraleiro-Pé-Duro bulls were used for cryopreservation. The cryopreserved spermatozoa were submitted to postthawmotility sperm of computer assisted analysis (CASA) to evaluate the characteristics of spermatic kinetics.Observed that The different concentrations of limonene (S) - (-) did not affect the parameters of kinetics spermaticexcept for linearity (LIN). The addition of 150 μM limonene (S) - (-) significantly increased (P <0.05) the LINcompared to the control. The results obtained in the present study allow us to conclude that the supplementationof limonene (S) - (-) in cryopreservation bovine semen diluent did not interfere in kinetics sperm.(AU)

Ação do Limoneno (S)-(-) sobre a cinética espermática de sêmen criopreservado de touros / Action of the Limonene (S) - (-) on sperm kinetics of cryopreserved semen of bulls

This study was conducted to evaluate the effect of different concentrations of Limoneno (S)-(-) (50 µM,100 µM and 150 µM) in supplementing the diluter of bulls freezing semen. Thirty-six ejaculated from fourCurraleiro-Pé-Duro bulls were used for cryopreservation. The cryopreserved spermatozoa were submitted to postthawmotility sperm of computer assisted analysis (CASA) to evaluate the characteristics of spermatic kinetics.Observed that The different concentrations of limonene (S) - (-) did not affect the parameters of kinetics spermaticexcept for linearity (LIN). The addition of 150 μM limonene (S) - (-) significantly increased (P <0.05) the LINcompared to the control. The results obtained in the present study allow us to conclude that the supplementationof limonene (S) - (-) in cryopreservation bovine semen diluent did not interfere in kinetics sperm.

Avaliação da integridade da membrana plasmática e do potencial de atividade mitocondrial de sêmen criopreservado de touros frente a ação do (Limoneno (R)-(+) / Evaluation of plasma membrane integrity and mitochondrial activity potential of cryopreserved semen of bulls on action of the Limonene (R) - (+)

This study was conducted to evaluate the effect of different concentrations of limonene (R)-(+) (50 µM,100 µM and 150 µM) in supplementing the diluter of bulls freezing semen. Thirty-six ejaculated from fourCurraleiro-Pé-Duro bulls were used for cryopreservation. The epifluorescence microscopy was used todetermine the plasmatic integrity and mitochondrial activity potential. No was observed effect on the integrity ofplasma membrane nor mitochondrial activity potential when cryopreserved by adding limonene R - (+). Theresults obtained in this study allow to conclude that supplementation limonene (R) - (+) on diluter of bullsfreezing semen does not interfere with the integrity of the plasma membrane or the potential of spermmitochondrial activity.

Efeito da melatonina sobre a integridade das membranas plasmática e mitocondrial de espermatozoides criopreservados de ovino / Effect of Melatonin on the plasmatic and mitochondrial integrity membrane of sperm cryopreserved ram

This study evaluate the effect of melatonin supplementation to TRIS-GEMA diluter in plasmatic andmitochondria membrane integrity of semen ram cryopreserved. Was used seven ejaculates of six ram,whichwere pooled in pool, according to the experimental groups: Control; T1 - 1x10-7 mol / L and T2 - 1x10-9 mol / Lmelatonin. Posteriorly the diluting the samples were packaged in straws of 0.25 ml and cryopreserved. Afterthawing at 37°C for 30 seconds, the samples were analyzed for Test thermoresistance slow (TTR) andacrosomal membrane integrity. For statistical analysis we used the Duncan test (α = 0.05) for meancomparison. Treatments 1 and 2 differed statistically control for plasma membrane integrity at times 60, 120and 180min. It was concluded that supplementation 1x10-7mol / L and 1x10-9mol / L of melatonin increased theintegrity of the plasma membrane to 60,120 and 180 minutes.

Efeito da melatonina sobre a viabilidade de espermatozóides criopreservados de ovinos / Effect of melatonin on the feasibility of sperm cryopreserved of ram

This study to evaluate the effect of melatonin supplementation to TRIS-GEMA diluter in sperm viabilityof semen ram cryopreserved. Was used seven ejaculates of six ram,which were pooled in pool, according to theexperimental groups: T1 - control; T2 - 1x10-7 mol / L and T3 - 1x10-9 mol / L melatonin. Posteriorly thediluting the samples were packaged in straws of 0.25 ml and cryopreserved. After thawing at 37°C for 30seconds, the samples were analyzed for Test thermoresistance slow (TTR) and acrosomal membrane integrity.For statistical analysis we used the Duncan test (α = 0.05) for mean comparison. The results demonstrated thatto the TTR at time 0 min, T2 showed 54.28% motility, and T1obtained lower values in the times 0 and 60 minutesfor force. Supplementation of 1x10-9mol / L of melatonin improved total motility.

INFLUÊNCIA DO TEMPO NA FERTILIDADE DO SÊMEN DESCONGELADO DE GARANHÕES DA RAÇA QUARTO DE MILHA

Quando se trata de biotecnologias da reprodução, é conhecido que o índice de fertilidade do sêmen congelado equino ainda é reduzido, uma vez que, os processos de congelação e descongelação levam a efeitos deletérios sobre o espermatozoide, diminuindo sua taxa de motilidade e vigor, modificando sua morfologia e, consequentemente, reduzindo o potencial fértil dos reprodutores. Por isso a necessidade de testes in vitro que avaliem a qualidade do sêmen equino descongelado, assim como sua viabilidade in vivo, levandose em consideração a possível influência da individualidade do animal sobre a forma de se comportar das células espermáticas frente aos processos de congelação e descongelação. Logo, o objetivo do presente estudo foi avaliar parâmetros de qualidade seminal in vitro de diferentes garanhões durante o teste de termorresistência, com o propósito de se obter uma técnica com maior repetibilidade, e que auxilie no incremento dos resultados no uso de sêmen congelado equino. Para isso, foram utilizadas amostras de sêmen de 4 garanhões da raça Quarto de Milha, em 5 repetições, submetidos a TTR nos tempos 0, 30, 60 e 90 minutos. A cinética espermática foi avaliada através do CASA. A integridade de membrana, foi avaliada pela coloração dupla (Diacetato de Carboxifluoresceína e Iodeto de Propídio), e a integridade do acrossoma, o Isotiocianeto de Fluoresceína conjugado a Peanut agglutin. A morfologia espermática foi analisada através de microscopia em câmara úmida com microscópio de contraste de fase. A viabilidade in vivo, foi testada pela inseminação de 10 éguas para gestação ou coleta de embriões distribuídos entre os reprodutores. Os dados foram analisados pelo Statistical Analysis System (SAS), com linearidade mista e parcelas repetidas no tempo, adotando covariância autorregressiva; e as médias comparadas pelo teste de Tukey Kramer. As correlações entre as variáveis foram avaliadas pelo coeficiente de correlação de Spearman. Foi observada uma interação significativa nos parâmetros Reprodutor, Tempo e interação reprodutor x tempo (p < 0,001) na cinética espermática, indicando individualidade entre garanhões em termos de longevidade espermática. Essa individualidade se repetiu na integridade de membrana, onde apenas um dos garanhões manteve membrana íntegra sem diferença significativa até os 90 minutos pós-TTR, enquanto que os demais reprodutores obtiveram diferença na integridade de membrana aos 30 e aos 60 minutos, respectivamente. Não houve diferença na patologia espermática ou na integridade de acrossoma entre tempos ou reprodutores. 50% das éguas inseminadas apresentaram embrião ou prenhez e todos as amostras seminais apresentaram pelo menos 1 égua prenhe ou embriões. Demonstrando nitidamente a individualidade dos reprodutores e a necessidade de mais conhecimento das características seminais de longevidade espermática dos mesmos, a fim de se padronizar protocolos de IA para poder determinar o momento mais adequado para a inseminação de acordo com o tempo da ovulação, mantendo-se índices de fertilidade melhores.

The aim of this study was to evaluate parameters of seminal quality in vitro of different stallions correlating with time, in order to obtain a technique with greater repeatability, and which helps to increase results in the use of frozen equine semen. Cryopreserved semen samples from 4 Quarter Horses stallions were used, in 5 repetitions, submitted to TTR at times 0, 30, 60 and 90 minutes. Sperm kinetics, membrane and acrosome integrity, sperm morphology were evaluated, and in vivo viability was demonstrated. Data analysis was performed by the Statistical Analysis System (SAS), in mixed linear models with plots repeated over time, adopting autoregressive covariance structure. And the means compared by the Tukey Kramer test. The correlations between the variables under study were assessed using Spearman's correlation coefficient. A significant interaction was observed in the parameters Breeder, Time and Breeder x time interaction (p <0.001) in sperm kinetics, indicating individuality between stallions in terms of sperm longevity. This individuality was repeated in the membrane integrity, where only one of the stallions maintained an intact membrane without significant difference until 90 minutes after TTR, while the other breeders obtained a difference in membrane integrity at 30 and 60 minutes, respectively. There was no difference in sperm pathology or acrosome integrity between times or reproducers. Of the inseminated mares 50% (5/10) presented embryo or pregnancy and all seminal samples presented at least 1 pregnant mare or produced embryos. The study clearly demonstrated the individuality of the breeders and the need for more knowledge of the semen characteristics of sperm longevity in order to standardize AI protocols taking into account the individuality of the stallion to determine the most appropriate time for insemination according to the time of ovulation, maintaining favorable fertility rates

Efeito da adição de caseinato de sódio sobre a viabilidade do sêmen bubalino criopreservado

O uso do sêmen refrigerado proporciona maiores taxas de prenhez se comparado ao do sêmen congelado. Essa diferença parece estar relacionada às lesões mais severas das membranas espermáticas desencadeadas pelo processo de congelação. Por sua habilidade de se ligar às proteínas ligadoras de espermatozoides e ao íon cálcio, o caseinato de sódio vem sendo estudado como uma substância capaz de inibir a capacitação espermática precoce, uma importante causa de diminuição da taxa de prenhez quando do uso de sêmen congelado. O primeiro objetivo deste estudo foi avaliar a possibilidade de um diluente comercial a base de gema de ovo, destinado à congelação de sêmen bovino, ser empregado para a criopreservação de sêmen bubalino; o segundo objetivo foi investigar o efeito do uso desse diluente, suplementado com caseinato de sódio, na criopreservação de espermatozoides bubalinos, por meio da avaliação dos espermatozoides, por citometria de fluxo, e da cinética espermática, empregando-se o sistema CASA. Na primeira parte do estudo, quando comparados os resultados das avaliações da cinética espermática e integridade das membranas plasmática e acrossomal, observou-se que o processo de congelação seminal promoveu mais danos celulares que o processo de refrigeração. Na segunda parte do estudo, não foram observados efeitos da adição do caseinato de sódio ao diluente a base de gema de ovo. A partir dos resultados do presente estudo foi possível concluir que o diluente a base de gema de ovo testado foi adequado para a criopreservação de sêmen bubalino e que a adição do caseinato de sódio não diminuiu os efeitos deletérios relacionados à criopreservação seminal.

The use of cooled semen results in higher pregnancy rates compared than the use of frozen semen. This result seems to be related to the more severe damages triggered by the freezing process, when compared to those observed during the refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a ubstance capable to prevent early sperm capacitation, a major cause of decreased pregnancy rate after using frozen semen. The first objective of this study was to evaluate if a commercial egg yolk diluent developed for freezing bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent, added with sodium caseinate, during the procedures of buffalo sperm cryopreservation, using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membranes integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the deleterious effects related to seminal cryopreservation.

Qualidade de oócitos caprinos após criopreservação em meio alternativo contendo ACP407®

Objetivou-se avaliar a viabilidade de oócitos caprinos submetidos ao processo de criopreservação lenta em meio alternativo contendo ACP-407® após descongelação. Foram coletados 160 ovários, provenientes de 80 cabras púberes oriundas de abatedouros localizados na microrregião homogênea de Teresina. Após o abateos ovários foram imediatamente transportados para o laboratório em garrafa térmica com solução salina fisiológica a 35°C adicionada de 30ug/mL de sulfato de gentamicina. Os oócitos foram aspirados com agulha 21G acoplada a seringa de 5mL, posteriormente o aspirado foi colocado em tubos tipo falconde 15 mL, onde permaneceram durante 15 minutos para que ocorresse a decantação dos Complexos CumulusOócitos (CCOs).O sedimento foi aspirado com o auxílio de uma pipeta de 100L e colocados em placa de petri onde foram avaliados e classificados utilizando estereomicroscópio. Após a classificaçãoos CCOs foram separados em três grupos, Grupo I (Glicerol - Controle), Grupo II (Etilenoglicol) e Grupo III (Etilenoglicol e ACP-407®), e envasados em palhetas francesas de 0,25 mL no qual foramsubmetidos ao processo de criopreservação lenta, em curva de criopreservaçãoespecifica para embriões,em aparelho TK 3000®. Após o período mínimo de 10 dias os oócitos foram descongelados e avaliados através da associação de sondas fluorescentes (iodeto de propídeoediacetato de carboxifluresceina).Foram recuperados 139 CCOs, onde obteve-se uma taxa de recuperação de 1,73CCOs por par de ovários. Em relação a análise de viabilidade com a utilização de sondas fluorescentes o grupo contendo ACP-407® cometilenoglicol apresentou melhores resultados quando comparados ao grupo etilenoglicol ou glicerol, representado uma taxa de viabilidade de 16,6%, 7,4% e 6%, respectivamente. Portanto, a adição de ACP-407® ao crioprotetoretilenoglicolmelhora a viabilidade dos oócitos caprinos pós descongelação.

The aim of this study was to evaluate the viability of goat oocytes submitted to the slow cryopreservation process in an alternative medium containing ACP-407® after thawing. A total of 160 ovaries were collected from 80 pre - pubescent goats from slaughterhouses located in the homogeneous microregion of Teresina. After slaughter the ovaries were immediately transported to the laboratory in a thermos flask with physiological saline solution at 35°C added with 30ug/mL gentamicin sulfate. The oocytes were aspirated with a 21G needle attached to a 5mL syringe, and the aspirate was placed in 15mL falcon tubes, where they remained for 15 minutes for the decantation of Cumulus Oocyte Complexes (COCs). The sediment was aspirated with the aid of a 100L pipette and placed in petri dish where they were evaluated and classified using a magnifying glass. After classification, the COCs were separated into three groups, Group I (Glycerol - Control), Group II (Ethylene Glycol) and Group III (Ethylene Glycol and ACP-407®), and packed in 0.25mL vats in which they were submitted to the slow cryopreservation, in specific curve for embryos In TK 3000® apparatus. After the minimum period of 10 days the oocytes were thawed and evaluated through the association of Fluorescent probes (propionate iodide andcarboxyfluorescein diacetate). 139 COCs were recovered, where a recovery rate of 1.73 COCs per pair of ovaries was obtained. In relation to the feasibility analysis with the use of fluorescent probes the group containing ACP-407®with Ethylene glycol presented better results when compared with the group Ethylene glycol or Glycerol, representing a viability rate of 16.6%, 7.4% and 6%, respectively. Therefore, the addition of ACP-407® to the cryoprotectant ethylene glycol improvement the viability of oocytes goats thawing.

EFEITO DA ADIÇÃO DE CICLODEXTRINA CARREGADA COM COLESTEROL NO EJACULADO SOBRE A QUALIDADE DO SÊMEN CONGELADO/DESCONGELADO DE TOUROS ADULTOS DA RAÇA NELORE

A criopreservação de sêmen bovino facilita a difusão e a disponibilidade de material genético a ser utilizado na inseminação artificial e produção de embriões. Durante o processo de criopreservação as membranas espermáticas são submetidas a uma variedade de injúrias, o que inclui estresse térmico, mecânico, químico e osmótico, que induzem a ocorrência de danos parcialmente irreversíveis ao espermatozoide, decorrentes de modificações da membrana plasmática, pela desestabilização da bicamada lipídica. A perda de colesterol da membrana plasmática de espermatozoides criopreservados pode causar uma capacitação prematura, reduzindo a viabilidade no trato reprodutivo feminino. A adição de colesterol pode ajudar a minimizar ou eliminar a transição de fase durante o processo de resfriamento. O colesterol pode ser facilmente incorporado ou extraído das membranas plasmáticas das células usando ciclodextrinas. As ciclodextrinas são oligossacarídeos cíclicos compostos por seis ( ciclodextrina), sete (- ciclodextrina) ou oito (- ciclodextrina) unidades de glicopiranose unidas por ligações - (1,4), cujas moléculas se caracterizam por ter uma superfície externa hidrofílica e uma cavidade interna lipofílica. Modificando as ciclodextrinas com grupos metil ou hidroxipropil aumenta-se a sua solubilidade em água e, consequentemente, a sua capacidade para solubilizar compostos hidrofóbicos. Neste experimento avaliou-se o efeito da adição de ciclodextrina carregada com colesterol (CCC) previamente ao processo de criopreservação do sêmen de touros adultos da raça Nelore. Foram utilizados cinco ejaculados de três touros. Cada ejaculado foi dividido em três frações: grupo controle e adição de 2 mg e 3 mg de CCC para cada 120x106 espermatozoides. As amostras congeladas/descongeladas foram avaliadas quanto aos aspectos físicos e morfológicos sob microscopia óptica e a cinética espermática pelo sistema CASA (Computer Assisted Sperm Analysis). Foram utilizados o teste hiposmótico e as colorações supravital e de epifluorescência para a avaliação da integridade da membrana plasmática dos espermatozoides congelados/descongelados. Não foram observadas diferenças significativas entre os grupos controle e tratados com CCC em relação aos aspectos físicos e morfológicos, parâmetros de cinética espermática (MT-%, MP-%, VSL-m/s, VCLm/s, VAP-m/s, RAP-%) e integridade de membrana plasmática dos espermatozoides (p>0,05). Conclui-se que a adição de CCC ao ejaculado de touros da raça Nelore previamente à criopreservação não melhora a qualidade do sêmen congelado/descongelado.

Cryopreservation of bovine semen facilitates the dissemination and availability of genetic material to be used in artificial insemination and embryo production. During the sperm cryopreservation membranes are subjected to a variety of insults, including thermal stress, mechanical, chemical and osmotic that induce the occurrence of partially irreversible damage to spermatozoa, due to modifications of the plasma membrane bilayer destabilization of the lipid. Cholesterol loss of plasma membrane of cryopreserved spermatozoa can cause premature capacitation, reducing the viability in the female reproductive tract. The addition of cholesterol can help to minimize or eliminate the phase transition during the cooling process. Cholesterol can be easily incorporated into or extracted from the plasma membranes of cells using cyclodextrins. Cyclodextrins are cyclic oligosaccharides consisting of six ( - cyclodextrin), seven (- cyclodextrin) or eight (- cyclodextrin) glucopyranose units linked by - (1-4) linked whose molecules are characterized by having an outer surface hydrophilic and a lipophilic inner cavity. Modifying cyclodextrins with hydroxypropyl methyl groups increases the water solubility and hence its ability to solubilize hydrophobic compounds. This experiment evaluated the effect of adding cyclodextrin loaded with cholesterol (CLC) prior to semen cryopreservation of adult Nelore bulls. Five ejaculates three bulls were used. Each ejaculate was divided into three fractions: control group and addition of 2 mg and 3 mg of CLC for each 120.106 sperm cells. Frozen/thawed samples were evaluated physical and morphology under light microscopy and sperm kinetics by CASA (Computer Assisted Sperm Analysis). Hiposmotic test and the supravital and epifluorescence stains were used to evaluation of membrane integrity of frozen/thawed spermatozoa. Significant differences between the control group and treated with CLC were not observed in relation to physical and morphological features, sperm kinetic parameters (MT-%, MP-%, VSL-m/s, VCLm/s, VAP-m/s, RAP-%) and plasma membrane integrity of spermatozoa (p> 0, 05). It is concluded that the addition of CCC to ejaculate of Nelore bulls prior to cryopreservation does not improve the quality of frozen / thawed semen.

CARACTERÍSTICAS FÍSICAS E MORFOLÓGICAS DO SÊMEN DE Bos taurus E Bos indicus ANTES E APÓS A CRIOPRESERVAÇÃO

O objetivo deste trabalho foi verificar as alterações que ocorrem nas células espermáticas durante o processo de criopreservação nas diferentes subespécies Bostaurus e Bosindicus. Analisou-se o ejaculado de 20 animais (10 de cada subespécie) quanto à motilidade, morfologia espermática, integridade de membrana plasmática e acrossoma, peroxidação lipídica, condensação da cromatina e morfometria da cabeça do espermatozoide no sêmen fresco e após criopreservação/descongelamento. Nas avaliações com sêmen fresco, a subespécie Bostaurus mostrou resultados de motilidade superior quando comparada aos zebuínos (68,00%±6,32% e 63,00%±4,83% respectivamente, p<0,05). Os animais europeus mostraram melhor congelabilidade do sêmen, pois seus espermatozoides, de maneira geral, foram menos afetados pelo processo de criopreservação (B. taurus Motilidade (%): 38,00±7,88, HIPO (%): 40,4±6,45, Área: 11556,14±2055,46, Perímetro: 25,17±1,87, Largura: 5,22±0,96, Fourier 2: 7867,27±1965,47; B. indicus - Motilidade: 30,00±4,71, HIPO: 27,5±6,98, Área: 12416,06±2232,06, Perímetro: 26,02±2,02, Largura: 5,31±0,97, Fourier 2: 8536,77±2058,21). Foi observado que a criopreservação causa danos em todas as estruturas espermáticas, independentemente da subespécie, indicando que avanços nesta técnica ainda são necessários.

This study aimed to verify the changes that occur in sperm cells during the process of cryopreservation between the different subspecies Bos taurus and Bos indicus. Semen from 20 animals (10 of each subspecies) fresh and after cryopreservation / thawing, were analyzed for motility, sperm morphology, plasma membrane and acrosome integrity, lipid peroxidation, chromatin condensation and sperm head morphology. The fresh semen evaluations results of the subspecies Bos taurus showed higher motility when compared to Zebu (68.00% ) 6.32% and 63.00% ) 4.83% respectively). European animals showed better freezability semen, in general, because their sperm were less affected by the cryopreservation process (B. taurus Motility (%): 38.00 ) 7.88, HYPO (%): 40.4 ) 6.45, Area : 11556.14 ) 2055.46, Perimeter: 25.17 ) 1.87, Width: 5.22 ) 0.96, Fourier 2: 7867.27 ) 1965.47;1 B. indicus - Motility: 30.00 ) 4.71, HYPO: 27.5 ) 6.98, area: 12416.06 ) 2232.06, Perimeter: 26.02 ) 2.02, Width: 5.31 ) 0.97, Fourier 2: 8536.77 ) 2058.21). It was observed that cryopreservation damages all sperm structures, regardless of subspecies, indicating that improvements in this technique are still needed.

Influence of Cooling Time and Diluents on the Freezability of Prochilodus brevis semen

Background: Prochilodus brevis is a rheophilic fish of economic and ecological importance. However, anthropic action has made its population vulnerable. Thus, the development of reproductive biotechnologies, such as seminal conservation, is necessary to subsidize their fish farming. However, seminal collections are often performed in places with few laboratory resources, demanding studies to determine the maximum time for which sperm can be cooled, as well as its process until frozen. Thus, the present study aimed to evaluate the influence of cooling time and the presence of dilution solutions on cryopreservation of P. brevis semen.Material, Methods & Results: After seminal collection, nine pools were formed and analyzed for seminal pH, concentration, membrane integrity, morphology and spermatic kinetics - motility, curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). After the analysis of the pools in natura (control 1), they were processed as follows: 1)- immediate freezing (control 2); 2)- cooling: undiluted, diluted in coconut water powder (ACP-104) or diluted in 5% glucose, followed by cooling at different times (6, 12, 24 or 48 h); 3)- Post-refrigeration freezing: the pools were diluted in their respective diluents and 10% dimethyl sulfoxide. After 15 days, the samples were thawed and analyzed for the aforementioned parameters. For

Influence of Cooling Time and Diluents on the Freezability of Prochilodus brevis semen

Background: Prochilodus brevis is a rheophilic fish of economic and ecological importance. However, anthropic action has made its population vulnerable. Thus, the development of reproductive biotechnologies, such as seminal conservation, is necessary to subsidize their fish farming. However, seminal collections are often performed in places with few laboratory resources, demanding studies to determine the maximum time for which sperm can be cooled, as well as its process until frozen. Thus, the present study aimed to evaluate the influence of cooling time and the presence of dilution solutions on cryopreservation of P. brevis semen.Material, Methods & Results: After seminal collection, nine pools were formed and analyzed for seminal pH, concentration, membrane integrity, morphology and spermatic kinetics - motility, curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). After the analysis of the pools in natura (control 1), they were processed as follows: 1)- immediate freezing (control 2); 2)- cooling: undiluted, diluted in coconut water powder (ACP-104) or diluted in 5% glucose, followed by cooling at different times (6, 12, 24 or 48 h); 3)- Post-refrigeration freezing: the pools were diluted in their respective diluents and 10% dimethyl sulfoxide. After 15 days, the samples were thawed and analyzed for the aforementioned parameters. For