Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.

Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.(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)

Utilização do choque osmótico na avaliação da viabilidade de sêmen criopreservado de ovinos / Utilization of the osmotic shock to assess the frozen ram semen viability

This study evaluated the use of hipoosmotic swelling test (HOST) with deionized water (0 mOsmol), as a method of post thaw ram semen evaluation and correlate their findings with different techniques of semen evaluation. Therefore, twenty semen samples of 20 different adult rams were assessed as for kinetic sperm parameters through computerized system (IVOS 12, Hamiton Thorn Biosciences, Beverly, MA, EUA) and subjective analysis. The sperm membranes viability was carried out by the association of fluorescent probes (propidium iodide, JC-1 and FITC-PSA). The structural integrity of the plasma membrane was also studied through supravital test with eosin and the functional integrity of membrane evaluated by doing the hipoosmotic swelling test with deionized water (0 mOsmol), in the following proportions: One part of semen for 10 (HOST 10), 50 (HOST 50) and 100 (HOST 100) parts of water. After semen dilution in the different proportions it was fixed in formalin-buffered saline and analyzed with regard to percentage of HOST reactive sperm (bent/coiled). The percentage of reaction obtained for HOST 10 (33,1%); HOST 50 (32,8%) and HOST 100 (31,8%) did not differ significantly. HOST 10 presented positive correlation with the plasma membrane integrity by the EOS (r = 0,80; p 0,05). Positive correlations between HOST 50 and HOST 100 with sperm subpopulation with membrane integrity by fluorescence were observed (r = 0,83 and r = 0,85; p 0,01). The findings suggest that the HOST with deionized water can provide additional information for post thawing ram sperm viability evaluation(AU)

O presente trabalho analisou o emprego do choque osmótico (HOST) com o uso da água deionizada (0 mOsmol), como método de avaliação do sêmen ovino descongelado e correlacionou os resultado obtidos com aqueles encontrados em diferentes técnicas de avaliação de sêmen. Para tanto, vinte amostras de sêmen criopreservado (20 reprodutores), foram avaliadas quanto aos parâmetros de cinética espermática pelo sistema computadorizado (IVOS 12, Hamilton Thorne Biosciences, Beverly, MA, EUA) e análise subjetiva. A análise da viabilidade das membranas espermáticas foi efetuada com a associação de sondas fluorescentes (PI, JC-1 e FITC-PSA). A integridade estrutural da membrana plasmática foi estudada com o teste supravital com eosina (EOS) e, por fim, a integridade funcional da membrana foi avaliada pelo choque osmótico, com a utilização da água deionizada nas seguintes proporções: uma parte de sêmen para 10 (HOST 10), 50 (HOST 50) e para 100 (HOST 100) partes de água. Após a diluição do sêmen, nas diferentes proporções, as amostras foram analisadas quanto ao percentual de espermatozoides reativos ao HOST. Os valores de positividade obtidos para o HOST 10 (33,1%), HOST 50 (32,8%) e HOST 100 (31,8%) não diferiram significativamente. O HOST 10 apresentou importante correlação positiva com a integridade da membrana plasmática pela EOS (r = 0,8; p 0,05). Os HOST 50 e 100 demonstraram correlações positivas com subpopulações espermáticas com membrana plasmática íntegra pela fluorescência (r = 0,83 e r = 0,85; p 0,01). Os resultados obtidos revelaram que o HOST com água deionizada pode fornecer informações complementares para a avaliação da viabilidade do sêmen ovino pós-descongelação(AU)