Avaliação morfológica do concentrado autólogo de plaquetas em cães sob refrigeração e criopreservação em DMSO 6% / [Morphological evaluation of the autologous platelet concentrate in dogs under refrigeration and criopreservation in 6% DMSO]

The morphological characteristics of the autologous platelet concentrate (APC) of 31 dogs were evaluated after cooling and freezing in 6% DMSO. Blood from the jugular vein of each patient was collected and centrifuged at 191g for six minutes to obtain APC. In the fresh sample, the platelet count, MPV, PDW and cell morphology were evaluated. Four samples of each animal were sent for storage, one refrigerated at 4°C for seven days, another for 30 days and two more stored in a freezer at -80°C in the same time interval, using 6% DMSO as cryoprotectant. The conserved samples were submitted to the same laboratory analysis as the fresh sample. There was a difference between fresh and preserved samples for platelet count, cell concentration, MPV and PDW (P<0.05), except in the 30-day refrigerated group, which showed severe morphological changes. In the frozen group for seven days, no difference was observed in the percentage of activation (P>0.05). The results obtained lead to the conclusion that cryopreservation with 6% DMSO at -80°C for seven days is a favorable option for the maintenance of platelet concentrations and the morphological characteristics of APC in dogs.(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.(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.

Efeito do DMSO e glicerol na criopreservação de células tronco mesenquimais derivadas de líquido amniótico de fetos caprinos / Effect of DMSO and glycerol in criopreservation of mesenquimial trunk cells derived from amniotic fluid of caprine fetus

O objetivo do estudo foi avaliar a viabilidade e taxa de proliferação de células-tronco mesenquimais (CTMs) derivadas do líquido amniótico (LA) após cultivo in vitro, e o efeito de dois agentes crioprotetores. Foram utilizadas 9 cabras prenhes. As amostras foram colhidas de fetos caprinos por laparotomia, e delas obtidos as (CTMs) e submetidas ao cultivo in vitro. Posteriormente, uma fração das células foi criopreservada em meio DMSO (dimetilsulfóxido) ou glicerol e vitrificadas para posterior avaliação da viabilidade. O meio DMSO promoveu melhores taxas de sobrevida celular preservando as características de pluripotencialidade e de replicação in vitro.

The objective of the study was to evaluate the viability and proliferation rate of mesenchymal stem cells (MTCs) derived from amniotic fluid (LA) after in vitro culture, and the effect of two cryoprotective agents. 9 pregnant goats were used. The samples were collected from goat fetuses by laparotomy, and from them (CTMs) were obtained and cultured in vitro. Subsequently, a fraction of the cells were cryopreserved in DMSO (dimethylsulfoxide) or glycerol medium and vitrified for further evaluation of viability. The DMSO medium promoted better cell survival rates while preserving the characteristics of pluripotency and replication in vitro.

Uso do extrato de Aloe vera na criopreservação do sêmen suíno / Use of Aloe vera extract in swine semen criopreservation

A gema de ovo é utilizada como agente crioprotetor em diluentes de congelação de sêmen visando à proteção contra o choque térmico, apesar de apresentar uma possibilidade de contaminação microbiológica. Autores buscam no Aloe vera, um meio alternativo para a conservação do sêmen. O objetivo foi avaliar o uso do crioprotetor Aloe vera na criopreservação do sêmen de suíno. Foram utilizados 25 ejaculados, coletados através da técnica da mão enluvada. As amostras foram diluídas no diluente de refrigeração (gema de ovo 10,0%, Aloe vera 10, 20 e 30%) a 17 °C, diluente de congelação (diluente de refrigeração + glicerol 6,0%) 5 °C e armazenados em palhetas. As palhetas foram avaliadas quanto ao vigor, motilidade, funcionalidade da membrana, vitalidade, integridade de acrossoma e na análise de sêmen assistida (CASA). Os dados foram avaliados quanto à normalidade, em seguida analisados utilizando ANOVA e comparações múltiplas, as análises foram realizadas com nível significância de 5,0%, utilizando o programa R3.4.0. Utilizou-se estatística descritiva para dados de vigor, motilidade e CASA. Na avaliação do sêmen descongelado para a funcionalidade da membrana, vitalidade e integridade de acrossoma, os maiores valores foram encontrados para as amostras com a gema de ovo (p0,05). Os dados de vigor, motilidade e CASA, das amostras utilizando o Aloe vera, apresentaram valores de 0,0. Os resultados mostraram que as diferentes concentrações de Aloe vera não exerceram o efeito crioprotetor desejado sobre a célula espermática, sendo assim, entende-se que são necessários maiores estudos sobre a sua ação na conservação de sêmen, bem como uma possível interação com os espermatozoides suínos...

Egg yolk is used as a cryoprotective agent in semen freezing diluents for protection against thermal shock, despite the possibility of microbiological contamination. Authors search for Aloe vera, an alternative medium for the conservation of semen. The objective was to evaluate the use of the cryoprotectant Aloe vera in the cryopreservation of swine semen. We used 25 ejaculates, collected through the gloved hand technique. The samples were diluted in the coolant diluent (10.0% egg yolk, Aloe vera 10, 20 and 30%) at 17 °C, freezing diluent (refrigerant diluent + glycerol 6.0%) at 5 °C and stored in vales. The vanes were evaluated for vigor, motility, membrane functionality, vitality, acrosome integrity and assisted semen analysis (CASA). The data were evaluated for normality, then analyzed using ANOVA and multiple comparisons, the analyzes were performed with a significance level of 5.0%, using the program R3.4.0. Descriptive statistics were used for data on vigor, motility and CASA. In the evaluation of thawed semen for membrane functionality, acrosome integrity and vitality, the highest values were found for the egg yolk samples (p0.05). The data of vigor, motility and CASA, of the samples using Aloe vera presented values of 0.0. The results showed that the different concentrations of Aloe vera did not exert the desired cryoprotective effect on the sperm cell, so it is understood that further studies are needed on its action on semen conservation, as well as a possible interaction with swine spermatozoa. On the other hand, it is believed that additional studies on the cooling curve of the porcine semen are still necessary, aiming at its cryopreservation.

Efeito do DMSO e glicerol na criopreservação de células tronco mesenquimais derivadas de líquido amniótico de fetos caprinos / Effect of DMSO and glycerol in criopreservation of mesenquimial trunk cells derived from amniotic fluid of caprine fetus

O objetivo do estudo foi avaliar a viabilidade e taxa de proliferação de células-tronco mesenquimais (CTMs) derivadas do líquido amniótico (LA) após cultivo in vitro, e o efeito de dois agentes crioprotetores. Foram utilizadas 9 cabras prenhes. As amostras foram colhidas de fetos caprinos por laparotomia, e delas obtidos as (CTMs) e submetidas ao cultivo in vitro. Posteriormente, uma fração das células foi criopreservada em meio DMSO (dimetilsulfóxido) ou glicerol e vitrificadas para posterior avaliação da viabilidade. O meio DMSO promoveu melhores taxas de sobrevida celular preservando as características de pluripotencialidade e de replicação in vitro.(AU)

The objective of the study was to evaluate the viability and proliferation rate of mesenchymal stem cells (MTCs) derived from amniotic fluid (LA) after in vitro culture, and the effect of two cryoprotective agents. 9 pregnant goats were used. The samples were collected from goat fetuses by laparotomy, and from them (CTMs) were obtained and cultured in vitro. Subsequently, a fraction of the cells were cryopreserved in DMSO (dimethylsulfoxide) or glycerol medium and vitrified for further evaluation of viability. The DMSO medium promoted better cell survival rates while preserving the characteristics of pluripotency and replication in vitro.(AU)

Uso do extrato de Aloe vera na criopreservação do sêmen suíno / Use of Aloe vera extract in swine semen criopreservation

A gema de ovo é utilizada como agente crioprotetor em diluentes de congelação de sêmen visando à proteção contra o choque térmico, apesar de apresentar uma possibilidade de contaminação microbiológica. Autores buscam no Aloe vera, um meio alternativo para a conservação do sêmen. O objetivo foi avaliar o uso do crioprotetor Aloe vera na criopreservação do sêmen de suíno. Foram utilizados 25 ejaculados, coletados através da técnica da mão enluvada. As amostras foram diluídas no diluente de refrigeração (gema de ovo 10,0%, Aloe vera 10, 20 e 30%) a 17 °C, diluente de congelação (diluente de refrigeração + glicerol 6,0%) 5 °C e armazenados em palhetas. As palhetas foram avaliadas quanto ao vigor, motilidade, funcionalidade da membrana, vitalidade, integridade de acrossoma e na análise de sêmen assistida (CASA). Os dados foram avaliados quanto à normalidade, em seguida analisados utilizando ANOVA e comparações múltiplas, as análises foram realizadas com nível significância de 5,0%, utilizando o programa R3.4.0. Utilizou-se estatística descritiva para dados de vigor, motilidade e CASA. Na avaliação do sêmen descongelado para a funcionalidade da membrana, vitalidade e integridade de acrossoma, os maiores valores foram encontrados para as amostras com a gema de ovo (p<0,05), e entre as amostras que utilizaram o Aloe vera, não foram encontradas diferenças significativas entre as diferentes concentrações testadas (p>0,05). Os dados de vigor, motilidade e CASA, das amostras utilizando o Aloe vera, apresentaram valores de 0,0. Os resultados mostraram que as diferentes concentrações de Aloe vera não exerceram o efeito crioprotetor desejado sobre a célula espermática, sendo assim, entende-se que são necessários maiores estudos sobre a sua ação na conservação de sêmen, bem como uma possível interação com os espermatozoides suínos...(AU)

Egg yolk is used as a cryoprotective agent in semen freezing diluents for protection against thermal shock, despite the possibility of microbiological contamination. Authors search for Aloe vera, an alternative medium for the conservation of semen. The objective was to evaluate the use of the cryoprotectant Aloe vera in the cryopreservation of swine semen. We used 25 ejaculates, collected through the gloved hand technique. The samples were diluted in the coolant diluent (10.0% egg yolk, Aloe vera 10, 20 and 30%) at 17 °C, freezing diluent (refrigerant diluent + glycerol 6.0%) at 5 °C and stored in vales. The vanes were evaluated for vigor, motility, membrane functionality, vitality, acrosome integrity and assisted semen analysis (CASA). The data were evaluated for normality, then analyzed using ANOVA and multiple comparisons, the analyzes were performed with a significance level of 5.0%, using the program R3.4.0. Descriptive statistics were used for data on vigor, motility and CASA. In the evaluation of thawed semen for membrane functionality, acrosome integrity and vitality, the highest values were found for the egg yolk samples (p<0.05), and among the samples that used Aloe vera, found significant differences between the different concentrations tested (p>0.05). The data of vigor, motility and CASA, of the samples using Aloe vera presented values of 0.0. The results showed that the different concentrations of Aloe vera did not exert the desired cryoprotective effect on the sperm cell, so it is understood that further studies are needed on its action on semen conservation, as well as a possible interaction with swine spermatozoa. On the other hand, it is believed that additional studies on the cooling curve of the porcine semen are still necessary, aiming at its cryopreservation.(AU)

Dimetilacetamida associada ou não ao glicerol para criopreservação de sêmen ovino / Dimethylacetamide associated or not to glycerol for criopreservation of sheep semen

O objetivo do presente estudo foi testar a dimetilacetamida (DMA) em diferentes concentrações, associada ou não ao glicerol (GL), sobre a viabilidade espermática do sêmen ovino congelado. Foram utilizados 10 ejaculados de dois carneiros adultos da raça Santa Inês. Os ejaculados foram divididos em sete grupos experimentais, respeitando o limite máximo de 5% de DMA, sendo eles: GL6%, DMA3%, GL5%+DMA1%, GL4%+DMA2%, GL3%+DMA3%, GL2%+DMA4%, GL1%+DMA5%. Os espermatozoides criopreservados nos diferentes tratamentos foram analisados quanto à cinética subjetiva, integridade estrutural da membrana plasmática (EOS), integridade funcional da membrana plasmática (CO) e morfologia espermática, observando defeitos totais (DT) e defeitos maiores (DM). A motilidade total (MT) e a progressiva (MP) pós-descongelação nos grupos GL5%+DMA1%; GL4%+DMA2% e GL3%+DMA3%, foram semelhantes (P>0,05) ao tratamento controle (GL6%). Destes, o diluidor GL4%+DMA2% foi o único que promoveu a manutenção da MT e MP pós-descongelação, quando comparado com o sêmen in natura (P>0,05). Não foram observadas diferenças significativas (P>0,05) para os parâmetros de EOS, CO, DT e DM nos diferentes grupos avaliados. A dimetilacetamida associada ao glicerol mostrou-se eficaz na manutenção da viabilidade espermática em ovinos, avaliada pós-descongelação. Entretanto, foi observado efeito deletério da DMA nas concentrações mais elevadas ou quando não esteve associada ao glicerol.

The objective of the present study was to test dimethylacetamide (DMA) at different concentrations, associated or not to glycerol (GL), on the sperm viability of frozen sheep semen. Ten ejaculates of two adult sheep of Santa Ines breed were used. The ejaculates were divided into seven experimental groups, respecting the maximum limit of 5% of DMA: GL6%, DMA3%, GL5%+DMA1%, GL4%+DMA2%, GL3%+DMA3%, GL2%+DMA4%, and GL1%+DMA5%. The sperm cryopreserved in the different treatments was analyzed based on the subjective kinetic, structural integrity of the plasma membrane (EOS), functional integrity of the plasma membrane (OS) and sperm morphology, observing total defects (TD) and major defects (MD). The post-thawed total mortality (TM) and progressive mortality (PM) in the GL5%+DMA1% groups; GL4%+DMA2% and GL3%+DMA3% were similar (P> 0.05) to the control treatment (GL6%). Of these, the diluent GL4%+DMA2% was the only one that promoted the maintenance of post-thawed TM and PM when compared to in natura semen (P> 0.05). No significant differences (P> 0.05) were observed for the EOS, OS, TD and MD parameters, in the different groups evaluated. Dimethylacetamide associated to glycerol were effective in maintaining sperm viability in post-thawed sheep semen. However, a deleterious effect of DMA was observed at the highest concentrations or when it was not associated with glycerol.

Dimetilacetamida associada ou não ao glicerol para criopreservação de sêmen ovino / Dimethylacetamide associated or not to glycerol for criopreservation of sheep semen

O objetivo do presente estudo foi testar a dimetilacetamida (DMA) em diferentes concentrações, associada ou não ao glicerol (GL), sobre a viabilidade espermática do sêmen ovino congelado. Foram utilizados 10 ejaculados de dois carneiros adultos da raça Santa Inês. Os ejaculados foram divididos em sete grupos experimentais, respeitando o limite máximo de 5% de DMA, sendo eles: GL6%, DMA3%, GL5%+DMA1%, GL4%+DMA2%, GL3%+DMA3%, GL2%+DMA4%, GL1%+DMA5%. Os espermatozoides criopreservados nos diferentes tratamentos foram analisados quanto à cinética subjetiva, integridade estrutural da membrana plasmática (EOS), integridade funcional da membrana plasmática (CO) e morfologia espermática, observando defeitos totais (DT) e defeitos maiores (DM). A motilidade total (MT) e a progressiva (MP) pós-descongelação nos grupos GL5%+DMA1%; GL4%+DMA2% e GL3%+DMA3%, foram semelhantes (P>0,05) ao tratamento controle (GL6%). Destes, o diluidor GL4%+DMA2% foi o único que promoveu a manutenção da MT e MP pós-descongelação, quando comparado com o sêmen in natura (P>0,05). Não foram observadas diferenças significativas (P>0,05) para os parâmetros de EOS, CO, DT e DM nos diferentes grupos avaliados. A dimetilacetamida associada ao glicerol mostrou-se eficaz na manutenção da viabilidade espermática em ovinos, avaliada pós-descongelação. Entretanto, foi observado efeito deletério da DMA nas concentrações mais elevadas ou quando não esteve associada ao glicerol.(AU)

The objective of the present study was to test dimethylacetamide (DMA) at different concentrations, associated or not to glycerol (GL), on the sperm viability of frozen sheep semen. Ten ejaculates of two adult sheep of Santa Ines breed were used. The ejaculates were divided into seven experimental groups, respecting the maximum limit of 5% of DMA: GL6%, DMA3%, GL5%+DMA1%, GL4%+DMA2%, GL3%+DMA3%, GL2%+DMA4%, and GL1%+DMA5%. The sperm cryopreserved in the different treatments was analyzed based on the subjective kinetic, structural integrity of the plasma membrane (EOS), functional integrity of the plasma membrane (OS) and sperm morphology, observing total defects (TD) and major defects (MD). The post-thawed total mortality (TM) and progressive mortality (PM) in the GL5%+DMA1% groups; GL4%+DMA2% and GL3%+DMA3% were similar (P> 0.05) to the control treatment (GL6%). Of these, the diluent GL4%+DMA2% was the only one that promoted the maintenance of post-thawed TM and PM when compared to in natura semen (P> 0.05). No significant differences (P> 0.05) were observed for the EOS, OS, TD and MD parameters, in the different groups evaluated. Dimethylacetamide associated to glycerol were effective in maintaining sperm viability in post-thawed sheep semen. However, a deleterious effect of DMA was observed at the highest concentrations or when it was not associated with glycerol.(AU)

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)

Espermatozoides epididimários: refrigeração e criopreservação / Epididymal spermatozoa: refrigeration and criopreservation

A recuperação de espermatozoides epididimários após a morte possibilita a utilização do material genético de reprodutores de interesse zootécnico que morrem inesperadamente e de espécimes selvagens ou ameaçadas de extinção. O período máximo após a morte pelo qual os espermatozoides permanecem viáveis varia conforme a temperatura de armazenamento dos epidídimos. A refrigeração e a congelação são os métodos utilizados para preservação espermática, contudo os espermatozoides epididimários apresentam peculiaridades em seus processos de preservação. A refrigeração de espermatozoides epididimários pode ser realizada in situ ou in vitro, sendo que estas duas formas de refrigeração apresentam vantagens e desvantagens inerentes às técnicas. Os espermatozoides epididimários apresentam maior resistência à variações de temperatura e osmolaridade que tornam os processos de preservação diferentes dos aplicados ao sêmen colhido em vagina artificial ou eletroejaculação. Esta revisão visa foi compilar trabalhos referentes à recuperação e preservação espermática epididimária.(AU)

The recovery of epididymal spermatozoa after death is used to avail genetic material of breeding herders of zootechnical interest who die unexpectedly and of wild or endangered species. The maximum post mortem period by which sperm remain viable varies with the storage temperature of the epididymis. Refrigeration and cryopreservation are methods for sperm preservation, however, epididymal spermatozoa present peculiarities in their preservation processes. The cooling of epididymal spermatozoa can be performed in situ or in vitro, these two forms of refrigeration have advantages and disadvantages inherent in the techniques. The epididymal spermatozoa have greater resistance to variations in temperature and osmolarity that make conservation processes different from those applied to semen collected in an artificial vagina or electroejaculation. This review aims to compile works referring to epididymal sperm retrieval and preservation.(AU)

Espermatozoides epididimários: refrigeração e criopreservação / Epididymal spermatozoa: refrigeration and criopreservation

A recuperação de espermatozoides epididimários após a morte possibilita a utilização do material genético de reprodutores de interesse zootécnico que morrem inesperadamente e de espécimes selvagens ou ameaçadas de extinção. O período máximo após a morte pelo qual os espermatozoides permanecem viáveis varia conforme a temperatura de armazenamento dos epidídimos. A refrigeração e a congelação são os métodos utilizados para preservação espermática, contudo os espermatozoides epididimários apresentam peculiaridades em seus processos de preservação. A refrigeração de espermatozoides epididimários pode ser realizada in situ ou in vitro, sendo que estas duas formas de refrigeração apresentam vantagens e desvantagens inerentes às técnicas. Os espermatozoides epididimários apresentam maior resistência à variações de temperatura e osmolaridade que tornam os processos de preservação diferentes dos aplicados ao sêmen colhido em vagina artificial ou eletroejaculação. Esta revisão visa foi compilar trabalhos referentes à recuperação e preservação espermática epididimária.

The recovery of epididymal spermatozoa after death is used to avail genetic material of breeding herders of zootechnical interest who die unexpectedly and of wild or endangered species. The maximum post mortem period by which sperm remain viable varies with the storage temperature of the epididymis. Refrigeration and cryopreservation are methods for sperm preservation, however, epididymal spermatozoa present peculiarities in their preservation processes. The cooling of epididymal spermatozoa can be performed in situ or in vitro, these two forms of refrigeration have advantages and disadvantages inherent in the techniques. The epididymal spermatozoa have greater resistance to variations in temperature and osmolarity that make conservation processes different from those applied to semen collected in an artificial vagina or electroejaculation. This review aims to compile works referring to epididymal sperm retrieval and preservation.

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)

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.

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 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.

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)