Does resveratrol preserve the integrity of the sperm membrane in goats after thawing? / O resveratrol preserva a integridade da membrana espermática de caprinos após descongelamento?

This study aimed to determine the effects of resveratrol in the trisaminomethane (TRIS)-egg yolk extender and its optimal inclusion level for goat semen cryopreservation. Five ejaculates of three Anglo Nubian goats were used, each divided into four 200 µL aliquots for use in four treatments: 0.00 (control), 0.04, 0.08 and 0.12 mg mL-¹ resveratrol in the TRIS-egg yolk extender. We evaluated progressive sperm motility and sperm vigor post-dilution, post-cooling, and post thawing; membrane integrity (HOST); and acrosomal integrity and performed a slow thermoresistance test (STT). The data were submitted to a regression analysis at a 5% probability. There was no difference in progressive motility or sperm vigor in the post-dilution (89.5, 89.0, 88.7 and 88.3, and 4.9, 5.0, 4.9, and 4.9) or post-cooling (81.0, 82.0, 83.0, and 78.3; and 4.3, 4.3, 4.2, and 4.2) experiments (P > 0.05), or in the complementary acrosomal integrity test (42.0, 47.4, 42.2 and 38.2) (P > 0.05). However, the motility and vigor parameters decreased linearly in the post-thaw phase, as well as during the 2 hours of incubation on STT (P < 0.05). These factors increased quadratically when resveratrol was added to HOST, to an optimal level of 0.039 mg mL-¹ resveratrol for a plasma membrane integrity of 52.55% (P < 0.05). The inclusion of resveratrol was effective in maintaining sperm viability; in particular, it was effective in maintaining plasmatic membrane integrity during the cryopreservation process up to 0.039 mg mL-1, meaning that it could be an alternative to conventionally used seminal extenders in goats.(AU)

O objetivo deste estudo foi determinar os efeitos e o melhor nível de inclusão de resveratrol em meio diluidor TRIS gema de ovo para criopreservação de sêmen caprino. Foram utilizados cinco ejaculados de três bodes da raça Anglo Nubiana. Cada ejaculado foi dividido em quatro alíquotas de 200 µL, compondo quatro tratamentos: 0,00 (controle); 0,04; 0,08 e 0,12mg mL-¹ de resveratrol no diluidor TRIS gema de ovo. Foram avaliadas a motilidade espermática progressiva e o vigor espermático pós diluição, pós-resfriamento e pós-descongelamento; integridade de membrana (HOST); integridade acrossomal e teste de termorresistência lento (TTR). Os dados foram submetidos à análise de regressão a 5% de probabilidade. Não houve diferença para a motilidade progressiva e o vigor espermático, respectivamente, na pós-diluição (89,5; 89,0; 88,7 e 88,3) e (4,9; 5,0; 4,9 e 4,9) e pós-resfriamento (81,0; 82,0; 83,0 e 78,3) e (4,3; 4,3; 4,2 e 4,2) (P < 0,05), assim como para o teste complementar integridade acrossomal (42,0; 47,4; 42,2 e 38,2) (P > 0,05). No entanto, os parâmetros motilidade e vigor reduziram linearmente no pós-descongelamento, assim como durante as 2 horas de incubação no TTR (P < 0,05). Houve efeito quadrático com a inclusão de resveratrol para HOST, apresentando um nível ótimo de 0,039mg mL-¹ de resveratrol para integridade de membrana plasmática de 52,55% (P <0,05). A inclusão de resveratrol foi eficiente na manutenção da viabilidade espermática; em especial, foi eficiente na manutenção da integridade da membrana plasmática durante o processo de criopreservação até o nível de 0,039mg mL-¹, podendo ser uma alternativa na composição dos diluidores seminais de caprinos.(AU)

Does resveratrol preserve the integrity of the sperm membrane in goats after thawing? / O resveratrol preserva a integridade da membrana espermática de caprinos após descongelamento?

This study aimed to determine the effects of resveratrol in the trisaminomethane (TRIS)-egg yolk extender and its optimal inclusion level for goat semen cryopreservation. Five ejaculates of three Anglo Nubian goats were used, each divided into four 200 µL aliquots for use in four treatments: 0.00 (control), 0.04, 0.08 and 0.12 mg mL-¹ resveratrol in the TRIS-egg yolk extender. We evaluated progressive sperm motility and sperm vigor post-dilution, post-cooling, and post thawing; membrane integrity (HOST); and acrosomal integrity and performed a slow thermoresistance test (STT). The data were submitted to a regression analysis at a 5% probability. There was no difference in progressive motility or sperm vigor in the post-dilution (89.5, 89.0, 88.7 and 88.3, and 4.9, 5.0, 4.9, and 4.9) or post-cooling (81.0, 82.0, 83.0, and 78.3; and 4.3, 4.3, 4.2, and 4.2) experiments (P > 0.05), or in the complementary acrosomal integrity test (42.0, 47.4, 42.2 and 38.2) (P > 0.05). However, the motility and vigor parameters decreased linearly in the post-thaw phase, as well as during the 2 hours of incubation on STT (P < 0.05). These factors increased quadratically when resveratrol was added to HOST, to an optimal level of 0.039 mg mL-¹ resveratrol for a plasma membrane integrity of 52.55% (P < 0.05). The inclusion of resveratrol was effective in maintaining sperm viability; in particular, it was effective in maintaining plasmatic membrane integrity during the cryopreservation process up to 0.039 mg mL-1, meaning that it could be an alternative to conventionally used seminal extenders in goats.

O objetivo deste estudo foi determinar os efeitos e o melhor nível de inclusão de resveratrol em meio diluidor TRIS gema de ovo para criopreservação de sêmen caprino. Foram utilizados cinco ejaculados de três bodes da raça Anglo Nubiana. Cada ejaculado foi dividido em quatro alíquotas de 200 µL, compondo quatro tratamentos: 0,00 (controle); 0,04; 0,08 e 0,12mg mL-¹ de resveratrol no diluidor TRIS gema de ovo. Foram avaliadas a motilidade espermática progressiva e o vigor espermático pós diluição, pós-resfriamento e pós-descongelamento; integridade de membrana (HOST); integridade acrossomal e teste de termorresistência lento (TTR). Os dados foram submetidos à análise de regressão a 5% de probabilidade. Não houve diferença para a motilidade progressiva e o vigor espermático, respectivamente, na pós-diluição (89,5; 89,0; 88,7 e 88,3) e (4,9; 5,0; 4,9 e 4,9) e pós-resfriamento (81,0; 82,0; 83,0 e 78,3) e (4,3; 4,3; 4,2 e 4,2) (P 0,05). No entanto, os parâmetros motilidade e vigor reduziram linearmente no pós-descongelamento, assim como durante as 2 horas de incubação no TTR (P < 0,05). Houve efeito quadrático com a inclusão de resveratrol para HOST, apresentando um nível ótimo de 0,039mg mL-¹ de resveratrol para integridade de membrana plasmática de 52,55% (P <0,05). A inclusão de resveratrol foi eficiente na manutenção da viabilidade espermática; em especial, foi eficiente na manutenção da integridade da membrana plasmática durante o processo de criopreservação até o nível de 0,039mg mL-¹, podendo ser uma alternativa na composição dos diluidores seminais de caprinos.

Efeito da adição de taurina sobre espermatozoides ovinos refrigerados a 5°C / Effect of taurine addiction on ram sperm cooled at 5ºC

O objetivo foi avaliar o efeito da adição de diferentes concentrações de taurina ao diluente de resfriamento de sêmen ovino. Foram utilizados neste experimento 40 ejaculados de oito carneiros, diluídos nostratamentos: controle Tris-gema (T1) adicionado de taurina nas concentrações de 1 µM (T2), 2 µM (T3) e 3 µM (T4), sob refrigeração a 5°C por 48 h e avaliados através da análise de parâmetros de qualidade espermática de integridade de membrana, DNA e acrossoma, funcionalidade de mitocôndria e motilidade espermática. Observou-se que a motilidade espermática nas 48 h foi inferior no T2 (29,3%) em relação ao T4 (37,6%) (P < 0,05). Quanto aos parâmetros de integridade de membrana e DNA não se verificou diferença estatística entre os tratamentos.Para integridade de acrossoma, em amostras de sêmen fresco, encontrou-se 59.0%, e após 48 h de refrigeração, foram observadas taxas integridade nos grupos adicionados de taurina de 50.7% (T2), 51,3% (T3) e 51.6% (T4),que não diferiram do sêmen fresco. Conclui-se que a taurina nas concentrações testadas foi eficiente para manter a integridade de acrossoma no sêmen ovino refrigerado a 5°C.(AU)

The objective of this work was to assess the effect of different concentrations of taurine added to ramsperm cooling extender. We used in this experiment 40 ejaculates from eight rams, diluted according to the following treatments: Tris-yolk control (T1) taurine-added at the concentrations of 1 µM (T2), 2 µM (T3) and 3 µM (T4), under 5º C refrigeration for 48 hours and assessed through sperm quality parameters of membrane integrity, DNA and acrosome, mitochondrial functionality and sperm motility. We observed that sperm motilitywithin 48 hours was lower in T2 (29.3%) when compared to T4 (37.6%) (P < 0.05). As for the parameters membrane integrity and DNA we did not observe statistical differences among the treatments. As for acrosome integrity, in fresh semen samples, we obtained 59.0%, and after 48 hours refrigeration we observed integrity rates in the taurine-added groups of 50.7% (T2), 51,3% (T3) and 51.6% (T4), that did not differ from fresh semen. In conclusion, the concentrations of taurine we tested was efficient to keep acrosome integrity within cooled ram sperm at 5ºC.(AU)

Efeito da adição de taurina sobre espermatozoides ovinos refrigerados a 5°C / Effect of taurine addiction on ram sperm cooled at 5ºC

O objetivo foi avaliar o efeito da adição de diferentes concentrações de taurina ao diluente de resfriamento de sêmen ovino. Foram utilizados neste experimento 40 ejaculados de oito carneiros, diluídos nostratamentos: controle Tris-gema (T1) adicionado de taurina nas concentrações de 1 µM (T2), 2 µM (T3) e 3 µM (T4), sob refrigeração a 5°C por 48 h e avaliados através da análise de parâmetros de qualidade espermática de integridade de membrana, DNA e acrossoma, funcionalidade de mitocôndria e motilidade espermática. Observou-se que a motilidade espermática nas 48 h foi inferior no T2 (29,3%) em relação ao T4 (37,6%) (P < 0,05). Quanto aos parâmetros de integridade de membrana e DNA não se verificou diferença estatística entre os tratamentos.Para integridade de acrossoma, em amostras de sêmen fresco, encontrou-se 59.0%, e após 48 h de refrigeração, foram observadas taxas integridade nos grupos adicionados de taurina de 50.7% (T2), 51,3% (T3) e 51.6% (T4),que não diferiram do sêmen fresco. Conclui-se que a taurina nas concentrações testadas foi eficiente para manter a integridade de acrossoma no sêmen ovino refrigerado a 5°C.

The objective of this work was to assess the effect of different concentrations of taurine added to ramsperm cooling extender. We used in this experiment 40 ejaculates from eight rams, diluted according to the following treatments: Tris-yolk control (T1) taurine-added at the concentrations of 1 µM (T2), 2 µM (T3) and 3 µM (T4), under 5º C refrigeration for 48 hours and assessed through sperm quality parameters of membrane integrity, DNA and acrosome, mitochondrial functionality and sperm motility. We observed that sperm motilitywithin 48 hours was lower in T2 (29.3%) when compared to T4 (37.6%) (P < 0.05). As for the parameters membrane integrity and DNA we did not observe statistical differences among the treatments. As for acrosome integrity, in fresh semen samples, we obtained 59.0%, and after 48 hours refrigeration we observed integrity rates in the taurine-added groups of 50.7% (T2), 51,3% (T3) and 51.6% (T4), that did not differ from fresh semen. In conclusion, the concentrations of taurine we tested was efficient to keep acrosome integrity within cooled ram sperm at 5ºC.

Avaliação da toxicidade de solvente de extratos vegetais com ação antiviral em sêmen caprino refrigerado / Evaluation of solvent toxicity of plant extract with antiviral action in refrigerated goat semen

Background: Caprine Arthritis Encephalitis Virus have been detected in sperm of breeding goats causing economic losses. In order to control the virus, researches aiming to identify natural extracts with potential antiviral effects are performed. However, aqueous or ethanolic extracts must be diluted in dimethyl sulfoxide (DMSO), which is a substance with unknown effects in sperm quality when present in diluting media. Therefore, this study aimed to evaluate sperm viability of refrigerated caprine semen diluted in media containing DMSO. This was performed to provide data that aid in researches involving the use of this component with natural extracts that may inactivate the caprine lentivirus in sperm.Materials, Methods & Results: The experiment was performed at the Laboratory of Seminal Technology in Embrapa Goats and Sheep in the city of Sobral, Brazil. Sperm viability was assessed in caprine semen refrigerated in two dilution media with crescent concentrations of DMSO. Sperm samples of five goats seronegative for the caprine lentivirus were pooled and diluted in minimal essential medium (MEM) enriched with glucose at 0.01 M added of crescent concentrations of DMSO (0%, 1.5%, 1.75%, 2.0%, 2.25% and 2.5%). The same breeders provided the pool of sperm to test Tris added 2.5% of egg yolk and the same concentrations of DMSO previously mentioned. Treatments were refrigerated at 7°C and evaluated up until four h after DMSO addition. Individual progressive motility (MIP), sperm vigor (V), percentage of spermatozoa reactive to hypoosmotic test (HO) and morphologically normal (NOR) were evaluated. IPM, vigor and NOR remained within normal standards for the caprine species in all treatments test. Percentage results of spermatozoa reactive to hypoosmotic was higher in Tris yolk with values ranging between 34.66% to 46.33%. Sperm vigor was positively correlated (r = 0.85) with IPM in the MEM diluted pool of sperm.[...]

Avaliação da toxicidade de solvente de extratos vegetais com ação antiviral em sêmen caprino refrigerado / Evaluation of solvent toxicity of plant extract with antiviral action in refrigerated goat semen

Background: Caprine Arthritis Encephalitis Virus have been detected in sperm of breeding goats causing economic losses. In order to control the virus, researches aiming to identify natural extracts with potential antiviral effects are performed. However, aqueous or ethanolic extracts must be diluted in dimethyl sulfoxide (DMSO), which is a substance with unknown effects in sperm quality when present in diluting media. Therefore, this study aimed to evaluate sperm viability of refrigerated caprine semen diluted in media containing DMSO. This was performed to provide data that aid in researches involving the use of this component with natural extracts that may inactivate the caprine lentivirus in sperm.Materials, Methods & Results: The experiment was performed at the Laboratory of Seminal Technology in Embrapa Goats and Sheep in the city of Sobral, Brazil. Sperm viability was assessed in caprine semen refrigerated in two dilution media with crescent concentrations of DMSO. Sperm samples of five goats seronegative for the caprine lentivirus were pooled and diluted in minimal essential medium (MEM) enriched with glucose at 0.01 M added of crescent concentrations of DMSO (0%, 1.5%, 1.75%, 2.0%, 2.25% and 2.5%). The same breeders provided the pool of sperm to test Tris added 2.5% of egg yolk and the same concentrations of DMSO previously mentioned. Treatments were refrigerated at 7°C and evaluated up until four h after DMSO addition. Individual progressive motility (MIP), sperm vigor (V), percentage of spermatozoa reactive to hypoosmotic test (HO) and morphologically normal (NOR) were evaluated. IPM, vigor and NOR remained within normal standards for the caprine species in all treatments test. Percentage results of spermatozoa reactive to hypoosmotic was higher in Tris yolk with values ranging between 34.66% to 46.33%. Sperm vigor was positively correlated (r = 0.85) with IPM in the MEM diluted pool of sperm.[...](AU)

Ácido ascórbico no diluidor para criopreservação de sêmen caprino / Ascorbic acid in extender for cryopreservation of goat semen

Objetivou-se determinar o efeito e o melhor nível de inclusão de ácido ascórbico em meio diluidor TRIS-Gema para criopreservação de sêmen caprino. Foram utilizados cinco ejaculados de três bodes da raça Anglo Nubiana. Cada ejaculado foi dividido em quatro alíquotas de 200 μL, compondo quatro tratamentos: um sem adição de ácido ascórbico (controle) e os demais com 0,0528; 0,1056 e 0,1584mg/mL de ácido ascórbico no diluidor TRIS-Gema. Avaliaram-se motilidade espermática progressiva e vigor espermático pós-diluição, pós-resfriamento e pós-descongelamento; integridade de membrana (HOST); integridade acrossomal e teste de termorresistência lento (TTR). Os dados foram submetidos à Análise de Regressão a 5% de probabilidade. Não houve diferença para motilidade progressiva e para vigor espermático, repectivamente, na pós-diluição; pós-resfriamento e pós-descongelamento, assim como para os testes complementares HOST e após 120 minutos do TTR, para motilidade e vigor, no pós-descongelamento (P>0,05). Houve comportamento quadrático com a inclusão do ácido ascórbico para integridade acrossomal (61,58%), com nível ótimo de 0,1006mg/mL de ácido ascórbico (P<0,05). A inclusão de 0,1006mg/mL de ácido ascórbico no diluidor TRIS-Gema melhorou a integridade acrossomal durante o processo de criopreservação, podendo ser uma alternativa na composição dos diluidores seminais caprinos.(AU)

The objective of this study was to determine the optimal inclusion level of ascorbic acid in TRIS-yolk extenders for cryopreservation of goat semen. We used five ejaculates of three Anglo Nubian goats. Semen from each animal was divided into four 200μL aliquots, composing four treatments: no addition of ascorbic acid (control) and 0.0528, 0.1056, and 0.1584mg/mL of ascorbic acid in TRIS-egg yolk. We evaluated motility and sperm vigor after dilution, post-cooling, and post-thawing; membrane integrity (HOST); acrosomal integrity, and slow thermoresistance test (TRT). Data were subjected to regression analysis at 5% probability. There was no difference for the progressive motility and sperm vigor, respectively, in the post-dilution, after cooling and post-thawing, as well as the additional tests HOST and after 120 minutes of TRT, for motility and force, respectively, in post-thawing (P>0.05). There was a quadratic effect with the addition of ascorbic acid to the acrosome integrity parameter, presenting a great maximum level of 0.1006 mg/mL ascorbic acid for acrosome integrity of 61.58% (P<0.05). The addition of ascorbic acid was effective in maintaining sperm viability, especially acrosome integrity during the cryopreservation process until the level of 0.1006 mg/mL, and it may be an alternative composition of seminal extenders for goats.(AU)

Ácido ascórbico no diluidor para criopreservação de sêmen caprino / Ascorbic acid in extender for cryopreservation of goat semen

Objetivou-se determinar o efeito e o melhor nível de inclusão de ácido ascórbico em meio diluidor TRIS-Gema para criopreservação de sêmen caprino. Foram utilizados cinco ejaculados de três bodes da raça Anglo Nubiana. Cada ejaculado foi dividido em quatro alíquotas de 200 μL, compondo quatro tratamentos: um sem adição de ácido ascórbico (controle) e os demais com 0,0528; 0,1056 e 0,1584mg/mL de ácido ascórbico no diluidor TRIS-Gema. Avaliaram-se motilidade espermática progressiva e vigor espermático pós-diluição, pós-resfriamento e pós-descongelamento; integridade de membrana (HOST); integridade acrossomal e teste de termorresistência lento (TTR). Os dados foram submetidos à Análise de Regressão a 5% de probabilidade. Não houve diferença para motilidade progressiva e para vigor espermático, repectivamente, na pós-diluição; pós-resfriamento e pós-descongelamento, assim como para os testes complementares HOST e após 120 minutos do TTR, para motilidade e vigor, no pós-descongelamento (P>0,05). Houve comportamento quadrático com a inclusão do ácido ascórbico para integridade acrossomal (61,58%), com nível ótimo de 0,1006mg/mL de ácido ascórbico (P<0,05). A inclusão de 0,1006mg/mL de ácido ascórbico no diluidor TRIS-Gema melhorou a integridade acrossomal durante o processo de criopreservação, podendo ser uma alternativa na composição dos diluidores seminais caprinos.

The objective of this study was to determine the optimal inclusion level of ascorbic acid in TRIS-yolk extenders for cryopreservation of goat semen. We used five ejaculates of three Anglo Nubian goats. Semen from each animal was divided into four 200μL aliquots, composing four treatments: no addition of ascorbic acid (control) and 0.0528, 0.1056, and 0.1584mg/mL of ascorbic acid in TRIS-egg yolk. We evaluated motility and sperm vigor after dilution, post-cooling, and post-thawing; membrane integrity (HOST); acrosomal integrity, and slow thermoresistance test (TRT). Data were subjected to regression analysis at 5% probability. There was no difference for the progressive motility and sperm vigor, respectively, in the post-dilution, after cooling and post-thawing, as well as the additional tests HOST and after 120 minutes of TRT, for motility and force, respectively, in post-thawing (P>0.05). There was a quadratic effect with the addition of ascorbic acid to the acrosome integrity parameter, presenting a great maximum level of 0.1006 mg/mL ascorbic acid for acrosome integrity of 61.58% (P<0.05). The addition of ascorbic acid was effective in maintaining sperm viability, especially acrosome integrity during the cryopreservation process until the level of 0.1006 mg/mL, and it may be an alternative composition of seminal extenders for goats.

Metil-formamida na criopreservação de sêmen ovino / Methyl-formamide in ram semen cryopreservation

Objetivou-se avaliar a eficácia da metil-formamida na criopreservação do sêmen ovino. O pool de sêmen utilizado no experimento foi obtido a partir da coleta, com vagina artificial, do sêmen de quatro carneiros mestiços Santa Inês, com idade aproximada de quatro anos. As coletas foram realizadas uma vez por semana, por seis semanas consecutivas, correspondendo, cada semana, a uma repetição do experimento. As frações do pool foram diluídas em cinco diferentes meios de congelação: (1) tris-gema com 5,3% de glicerol (TG5,3G); (2) tris-gema com 3% de metil-formamida (TG3MF); (3) tris-gema com 5% de metil-formamida (TG5MF); (4) tris-gema com 7% de metil-formamida (TG7MF); (5) tris-gema com 9% de metil-formamida (TG9MF). Foram avaliadas a motilidade progressiva e o vigor das células espermáticas e realizado o teste de termorresistência pós-descongelação. O tratamento que obteve maior motilidade foi o TG5,3G (50%), seguido do TG3MF (38%) e os tratamentos que apresentaram menor motilidade progressiva foram TG5MF (29%), TG7MF (1,0%), TG9MF (6,0%). Os meios contendo metil-formamida apresentaram resultados inferiores ao meio controle para preservar a integridade morfológica dos espermatozoides, sendo que nos meios TG7MF e TG9MF menos de 60% de espermatozoides apresentaram-se morfologicamente normais. Os espermatozoides do meio TG5,3G  apresentaram motilidade (15%) e vigor (2,8) similares aos do meio TG3MF (15% e 2,6, respectivamente) no teste de termorresistência, mas o meio TG5,3G preservou melhor a integridade funcional da membrana plasmática. O glicerol foi mais eficiente como crioprotetor do que a metil-formamidana criopreservação de sêmen ovino.(AU)

The aim of this study was to evaluate the efficacy of methyl-formamide in ram semen cryopreservation. The semen pool used in this experiment was obtained by artificial vagina collection from four mixed breed Santa Inês rams, around four years of age. Semen collection was performed once a week, during six weeks. Each week corresponded to one experiment replication. The semen pool was divided in five fractions in order to be diluted in one of the following freezing media: (1) tris-egg yolk with 5.3% of glycerol (TG5.3G); (2) tris-egg yolk with 3% of methyl-formamide (TG3MF); tris-egg yolk with 5% of methyl-formamide (TG5MF); tris-egg yolk with 7% of methyl-formamide (TG7MF); tris-egg yolk with 9% of methyl-formamide (TG9MF). Semen progressive motility, vigor and thermoresistance were evaluated. The treatments TG5.3G (50%) and TG3MF (38%) showed higher progressive motility after thawing, while TG5MF (29%), TG7MF (1%) and TG9MF (6%) showerd lower motility. Freezing media containing methyl-formamide were less effective in preserving spermatozoa membrane integrity and morphology than control media. In TG7MF and TG9MF extenders, less than 60% spermatozoa showed normal morphology. After thermoresistance test, semen cryopreserved in TG3MF showed vigor (2.6) and motility (15%) statistically similar to TG5.3G media (15% and 2.8, respectively); however, the extender TG5.3G was more effective in preserving plasma membrane functional integrity. In conclusion, in the experimental conditions used, glycerol showed more cryoprotectant potential than methyl-formamide.(AU)

Metil-formamida na criopreservação de sêmen ovino / Methyl-formamide in ram semen cryopreservation

Objetivou-se avaliar a eficácia da metil-formamida na criopreservação do sêmen ovino. O pool de sêmen utilizado no experimento foi obtido a partir da coleta, com vagina artificial, do sêmen de quatro carneiros mestiços Santa Inês, com idade aproximada de quatro anos. As coletas foram realizadas uma vez por semana, por seis semanas consecutivas, correspondendo, cada semana, a uma repetição do experimento. As frações do pool foram diluídas em cinco diferentes meios de congelação: (1) tris-gema com 5,3% de glicerol (TG5,3G); (2) tris-gema com 3% de metil-formamida (TG3MF); (3) tris-gema com 5% de metil-formamida (TG5MF); (4) tris-gema com 7% de metil-formamida (TG7MF); (5) tris-gema com 9% de metil-formamida (TG9MF). Foram avaliadas a motilidade progressiva e o vigor das células espermáticas e realizado o teste de termorresistência pós-descongelação. O tratamento que obteve maior motilidade foi o TG5,3G (50%), seguido do TG3MF (38%) e os tratamentos que apresentaram menor motilidade progressiva foram TG5MF (29%), TG7MF (1,0%), TG9MF (6,0%). Os meios contendo metil-formamida apresentaram resultados inferiores ao meio controle para preservar a integridade morfológica dos espermatozoides, sendo que nos meios TG7MF e TG9MF menos de 60% de espermatozoides apresentaram-se morfologicamente normais. Os espermatozoides do meio TG5,3G  apresentaram motilidade (15%) e vigor (2,8) similares aos do meio TG3MF (15% e 2,6, respectivamente) no teste de termorresistência, mas o meio TG5,3G preservou melhor a integridade funcional da membrana plasmática. O glicerol foi mais eficiente como crioprotetor do que a metil-formamidana criopreservação de sêmen ovino.

The aim of this study was to evaluate the efficacy of methyl-formamide in ram semen cryopreservation. The semen pool used in this experiment was obtained by artificial vagina collection from four mixed breed Santa Inês rams, around four years of age. Semen collection was performed once a week, during six weeks. Each week corresponded to one experiment replication. The semen pool was divided in five fractions in order to be diluted in one of the following freezing media: (1) tris-egg yolk with 5.3% of glycerol (TG5.3G); (2) tris-egg yolk with 3% of methyl-formamide (TG3MF); tris-egg yolk with 5% of methyl-formamide (TG5MF); tris-egg yolk with 7% of methyl-formamide (TG7MF); tris-egg yolk with 9% of methyl-formamide (TG9MF). Semen progressive motility, vigor and thermoresistance were evaluated. The treatments TG5.3G (50%) and TG3MF (38%) showed higher progressive motility after thawing, while TG5MF (29%), TG7MF (1%) and TG9MF (6%) showerd lower motility. Freezing media containing methyl-formamide were less effective in preserving spermatozoa membrane integrity and morphology than control media. In TG7MF and TG9MF extenders, less than 60% spermatozoa showed normal morphology. After thermoresistance test, semen cryopreserved in TG3MF showed vigor (2.6) and motility (15%) statistically similar to TG5.3G media (15% and 2.8, respectively); however, the extender TG5.3G was more effective in preserving plasma membrane functional integrity. In conclusion, in the experimental conditions used, glycerol showed more cryoprotectant potential than methyl-formamide.