Effects of trehalose and catalase on the viability and kinetic parameters of cryopreserved ram sperm

Background: Most part of ram spermatozoa membrane has unsaturated fatty acids (phospholipids). Membrane structure of cells is composed of double ordered phospholipid layers adorned with mosaic-like protein, glycoprotein and glycolipids. Sperm freezing protocols could be negatively affected on ram sperm motility, viability and acrosome integrity during cryopreservation. For these reasons, researchers were designed their topics has led to the search for effective antioxidant systems against peroxidative damage and spermatozoon dysfunction. There are three protective enzymatic systems against reactive oxygen species (ROS) damage in sperm. These include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase / reductase cycles. Catalase is a hemo-protein in the enzyme tetramer structure. The aim of this study was to investigate the effects of trehalose, catalase and their combinations on ram sperm parameters after the cryopreservation/ thawing process.Materials, Methods & Results: At the out of breeding season (March-May) seven rams (1-3 years of age) were used in this study. Ejaculates were collected by electro-ejaculator twice a week. Pooled ejaculates were kept at 37°C, divided into six aliquots, diluted with the Tris based extender containing Trehalose 25 mM (Group-1), Trehalose 50 mM (Group-2), Catalase 200 µg (Group-3), Catalase 400 µg (Group-4), Trehalose 50 mM + Catalase 400 µg (Group-5) and no anti-oxidant (control), respectively, were cooled to 5°C than frozen in 0.25 mL French straws on the nitrogen vapour and stored in liquid nitrogen. The extender supplemented with Group 1 (54.1 ± 1.53; 73.1 ± 4.37), Group 2 (58.3 ± 4.01; 63.1 ± 0.30) and Group 5 (56.6 ± 1.05; 58.3 ± 0.55) resulted in higher subjective motility in comparison to the control (40.0 ± 3.87; 40.5 ± 0.22) group respectively (P < 0.05).[...]

Effects of trehalose and catalase on the viability and kinetic parameters of cryopreserved ram sperm

Background: Most part of ram spermatozoa membrane has unsaturated fatty acids (phospholipids). Membrane structure of cells is composed of double ordered phospholipid layers adorned with mosaic-like protein, glycoprotein and glycolipids. Sperm freezing protocols could be negatively affected on ram sperm motility, viability and acrosome integrity during cryopreservation. For these reasons, researchers were designed their topics has led to the search for effective antioxidant systems against peroxidative damage and spermatozoon dysfunction. There are three protective enzymatic systems against reactive oxygen species (ROS) damage in sperm. These include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase / reductase cycles. Catalase is a hemo-protein in the enzyme tetramer structure. The aim of this study was to investigate the effects of trehalose, catalase and their combinations on ram sperm parameters after the cryopreservation/ thawing process.Materials, Methods & Results: At the out of breeding season (March-May) seven rams (1-3 years of age) were used in this study. Ejaculates were collected by electro-ejaculator twice a week. Pooled ejaculates were kept at 37°C, divided into six aliquots, diluted with the Tris based extender containing Trehalose 25 mM (Group-1), Trehalose 50 mM (Group-2), Catalase 200 µg (Group-3), Catalase 400 µg (Group-4), Trehalose 50 mM + Catalase 400 µg (Group-5) and no anti-oxidant (control), respectively, were cooled to 5°C than frozen in 0.25 mL French straws on the nitrogen vapour and stored in liquid nitrogen. The extender supplemented with Group 1 (54.1 ± 1.53; 73.1 ± 4.37), Group 2 (58.3 ± 4.01; 63.1 ± 0.30) and Group 5 (56.6 ± 1.05; 58.3 ± 0.55) resulted in higher subjective motility in comparison to the control (40.0 ± 3.87; 40.5 ± 0.22) group respectively (P < 0.05).[...](AU)

Effect of the type of egg yolk, removal of seminal plasma and donor age on ram spermcryopreservation

This study was conducted to assess the effect on ram sperm freezability by the replacement of traditional fresh egg yolk (FEY) with fresh clarified egg yolk (CEY) or powdered egg yolk (PEY) in extenders. Simultaneously, the effect of semen washing and donor age (1 and 2 years old) was studied on thawed sperm quality. Briefly, in two consecutives autumns, ejaculates from 8 males were collected by artificial vagina, mixed and split into two samples. One sample was washed by centrifugation and the pellet was split into three aliquots and re-suspended in an extender containing 15% of different type of egg yolk (PEY, FEY or CEY) supplemented with 5% glycerol in a Tris-based medium. The other sample was directly split into three aliquots and diluted in the same extenders. All samples were chilled for 4 h at 5ºC before been frozen to −196ºC. The results suggested that powdered egg yolk can be used satisfactory on ram sperm cryopreservation ensuring greater bio-security meanwhile fresh clarified egg yolk did not improve sperm freezability. Moreover, semen from 2 years old donors was more resistant to cryopreservation than semen collected from younger males. Finally, sperm washing had a beneficial effect on sperm cryosurvival.

Effect of the type of egg yolk, removal of seminal plasma and donor age on ram spermcryopreservation

This study was conducted to assess the effect on ram sperm freezability by the replacement of traditional fresh egg yolk (FEY) with fresh clarified egg yolk (CEY) or powdered egg yolk (PEY) in extenders. Simultaneously, the effect of semen washing and donor age (1 and 2 years old) was studied on thawed sperm quality. Briefly, in two consecutives autumns, ejaculates from 8 males were collected by artificial vagina, mixed and split into two samples. One sample was washed by centrifugation and the pellet was split into three aliquots and re-suspended in an extender containing 15% of different type of egg yolk (PEY, FEY or CEY) supplemented with 5% glycerol in a Tris-based medium. The other sample was directly split into three aliquots and diluted in the same extenders. All samples were chilled for 4 h at 5ºC before been frozen to −196ºC. The results suggested that powdered egg yolk can be used satisfactory on ram sperm cryopreservation ensuring greater bio-security meanwhile fresh clarified egg yolk did not improve sperm freezability. Moreover, semen from 2 years old donors was more resistant to cryopreservation than semen collected from younger males. Finally, sperm washing had a beneficial effect on sperm cryosurvival.(AU)

Effect of L-arginine treatment on motility, hyperactivity, acrosome reaction of ejaculated ram spermatozoa

The aim of this study was to evaluate the effect of in vitro treatment of ejaculated ram spermatozoa with different concentrations of L-arginine at various incubation times on motility, hyperactivity (HA) and acrosome reaction. Freshly ejaculated spermatozoa collected from three rams were pooled and subjected to the swim up technique in modified sperm Tyrode's albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of Larginine (0.01, 0.02, 0.03,0.04 and 0.05 mM) at 30, 60,90 and 120 min of incubation. Following sperm incubation, the following parameters were examined: motility, hyperactivity (HA) and acrosome reaction (AR). The results showed that irrespective of the concentration, incubation of ram spermatozoa with Larginine for 30min did not significantly affect the motility. However, increase the time of incubation for more than 30 min significantly decreased (P < 0.05) the motility of the spermatozoa as compared to the control. The lowest motility was recorded when spermatozoa were incubated with 0.05 mM L-arginine for 120 min. Treatment of ram spermatozoa with 0.05 mM Larginine resulted in a significant (P < 0.05) increase in HA% immediately after dilution compared to the control. A significant (P < 0.05) increase in total AR% was concomitant to the increase in the concentration of L-arginine with highest AR achieved at 0.04 mM and 90 min incubation. However, increasing the time of incubation to 120 min significantly decreased (P < 0.05) the percentage of spermatozoa with total AR compared to the other incubation times at 0.02, 0.04, and 0.05 mM L-arginine. In conclusion, under our experimental conditions treatment of ejaculated ram spermatozoa with 0.04 mM of L-arginine for 90 min was considered the best concentration of L-arginine to be used for in vitro induction of acrosome reaction.

Effect of L-arginine treatment on motility, hyperactivity, acrosome reaction of ejaculated ram spermatozoa

The aim of this study was to evaluate the effect of in vitro treatment of ejaculated ram spermatozoa with different concentrations of L-arginine at various incubation times on motility, hyperactivity (HA) and acrosome reaction. Freshly ejaculated spermatozoa collected from three rams were pooled and subjected to the swim up technique in modified sperm Tyrode's albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of Larginine (0.01, 0.02, 0.03,0.04 and 0.05 mM) at 30, 60,90 and 120 min of incubation. Following sperm incubation, the following parameters were examined: motility, hyperactivity (HA) and acrosome reaction (AR). The results showed that irrespective of the concentration, incubation of ram spermatozoa with Larginine for 30min did not significantly affect the motility. However, increase the time of incubation for more than 30 min significantly decreased (P < 0.05) the motility of the spermatozoa as compared to the control. The lowest motility was recorded when spermatozoa were incubated with 0.05 mM L-arginine for 120 min. Treatment of ram spermatozoa with 0.05 mM Larginine resulted in a significant (P < 0.05) increase in HA% immediately after dilution compared to the control. A significant (P < 0.05) increase in total AR% was concomitant to the increase in the concentration of L-arginine with highest AR achieved at 0.04 mM and 90 min incubation. However, increasing the time of incubation to 120 min significantly decreased (P < 0.05) the percentage of spermatozoa with total AR compared to the other incubation times at 0.02, 0.04, and 0.05 mM L-arginine. In conclusion, under our experimental conditions treatment of ejaculated ram spermatozoa with 0.04 mM of L-arginine for 90 min was considered the best concentration of L-arginine to be used for in vitro induction of acrosome reaction.(AU)

Effect of ß-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos / Efeito do ß-mercaptoetanol e da cisteína sobre a qualidade e a atividade oxidativa do sêmen ovino após o descongelamento e sobre a viabilidade de embriões ovinos vitrificados

The effects of ß-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50µM BME and 600µM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.(AU)

Foram avaliados os efeitos do ß-mercaptoetanol (BME) e da cisteína sobre a viabilidade e a atividade oxidativa após o descongelamento do sêmen ovino e sobre o desenvolvimento in vitro e a viabilidade de embriões ovinos vitrificados. Ejaculados de quatro carneiros foram agrupados e diluídos, compondo seis tratamentos: sem antioxidantes; com BME 2mM; com BME 5mM; com BME 2mM e cisteína 5mM; com BME 5mM e cisteína 5mM; e com cisteína 5mM. Motilidade, integridade da membrana e do acrossoma, função mitocondrial, produção de espécies reativas de oxigênio e capacidade antioxidante total foram semelhantes entre os tratamentos (P>0,05). Em um meio sem antioxidantes, as taxas de clivagem e de desenvolvimento embrionário até blastocisto (60,3%, e 33,6%, respectivamente) foram semelhantes (P>0,05) às obtidas em um meio com BME 50µM e cisteína 600µM (64,3% e 36,6%, respectivamente). A viabilidade pós-descongelamento dos embriões vitrificados não diferiu entre os meios (P>0,05). O BME e a cisteína não influenciaram a viabilidade e a atividade oxidativa do sêmen ovino após o descongelamento e a viabilidade de embriões ovinos vitrificados.(AU)

Effect of ß-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos / Efeito do ß-mercaptoetanol e da cisteína sobre a qualidade e a atividade oxidativa do sêmen ovino após o descongelamento e sobre a viabilidade de embriões ovinos vitrificados

The effects of ß-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50µM BME and 600µM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.(AU)

Foram avaliados os efeitos do ß-mercaptoetanol (BME) e da cisteína sobre a viabilidade e a atividade oxidativa após o descongelamento do sêmen ovino e sobre o desenvolvimento in vitro e a viabilidade de embriões ovinos vitrificados. Ejaculados de quatro carneiros foram agrupados e diluídos, compondo seis tratamentos: sem antioxidantes; com BME 2mM; com BME 5mM; com BME 2mM e cisteína 5mM; com BME 5mM e cisteína 5mM; e com cisteína 5mM. Motilidade, integridade da membrana e do acrossoma, função mitocondrial, produção de espécies reativas de oxigênio e capacidade antioxidante total foram semelhantes entre os tratamentos (P>0,05). Em um meio sem antioxidantes, as taxas de clivagem e de desenvolvimento embrionário até blastocisto (60,3%, e 33,6%, respectivamente) foram semelhantes (P>0,05) às obtidas em um meio com BME 50µM e cisteína 600µM (64,3% e 36,6%, respectivamente). A viabilidade pós-descongelamento dos embriões vitrificados não diferiu entre os meios (P>0,05). O BME e a cisteína não influenciaram a viabilidade e a atividade oxidativa do sêmen ovino após o descongelamento e a viabilidade de embriões ovinos vitrificados.(AU)

EFEITO IN VITRO DO FLUIDO E CÉLULAS EPITELIAIS DO OVIDUTO BOVINO NA FASE FOLICULAR OU LUTEAL NA FUNÇÃO E CAPACITAÇÃO DE ESPERMATOZOIDES OVINOS

O fluido do oviduto (FO) desempenha importante função na capacitação espermática e fertilização. Ainda, as células epiteliais do oviduto (CEO) interagem com o espermatozoide (SPZ) prolongando a sua vida útil. Evidências na literatura sugerem que o FO varia de acordo com a fase do ciclo estral, no entanto, pouco se sabe sobre os efeitos dessa variação na função espermática. Assim, o objetivo do estudo foi avaliar o efeito do FO da fase folicular e luteal e a interação do SPZ com CEO na modulação da função espermática e dos processos de capacitação/reação acrossômica (RA). Para alcançar esse objetivo, dois experimentos foram realizados. Para isso, FO na fase folicular e luteal (classificação baseada na morfologia ovariana) e CEO foram obtidas de ovidutos de vacas oriundas de matadouro. Para ambos os experimentos, após a técnica de swim-up (pool de três carneiros), os SPZ foram distribuídos aleatoriamente nos grupos experimentais. No experimento 1, 8 x 106 SPZ/mL foram adicionados ao meio: i) Fert-TALP (controle positivo), ii) Fert-TALP sem indutores da capacitação espermática (Fert-TALP modificado; controle negativo), iii) Fert-TALP modificado, suplementado com 10% de FO da fase folicular ou iv) FertTALP modificado, suplementado com 10% de FO na fase luteal. No experimento 2, para mimetizar as condições do oviduto durante a fase folicular e luteal, as CEO foram cultivadas no meio TCM-199, suplementada com 10% de soro fetal bovino até a confluência. Previamente (24 h) ao cocultivo com SPZ, foi realizada a indução das condições luteal e folicular pela suplementação dos hormônios 17 -estradiol (E2) e progesterona (P4) no meio nas concentrações detectadas no oviduto durante estas fases. Para o cocultivo, 8 x 106 SPZ/mL foram adicionados na presença da monocamada de CEO: i) CEO fase folicular cocultivo de SPZ e CEO no meio FertTALP, com suplementação de 290 pg/mL E2 e 6 ng/mL P4 (concentração similar a fase folicular); ii) CEO fase luteal cocultivo de SPZ e CEO no meio Fert-TALP, com suplementação de 80 pg/mL E2 e 85 ng/mL P4 (concentração similar a fase luteal); iii) cultivo de SPZ no meio Fert-TALP, com suplementação de 290 pg/mL de E2 e 6 ng/mL P4 e iv) cultivo de SPZ no meio Fert-TALP, com suplementação de 80 pg/mL E2 e 85 ng/mL P4. Para o grupo controle positivo, foi utilizado o meio Fert-TALP convencional. Os SPZ foram incubados a 38 °C em 5% de CO2 e os parâmetros da cinemática espermática, integridade da membrana plasmática (MP) e status da capacitação espermática foram avaliados após 0, 2, 4, 6 e 18 e 24 h. As variáveis foram submetidas ao teste two-way ANOVA com medidas repetidas (Bonferroni; P < 0,05). Os resultados do Experimento 1, mostraram que a incubação de até 4 h com FO independente do estágio do ciclo estral promoveu aumento na maioria dos parâmetros da cinemática espermática de forma similar (P 0,05) ao grupo controle positivo, enquanto que no Experimento 2, esses parâmetros foram reduzidos (P < 0,05) pelo co-cultivo entre SPZ e CEO, independente do estágio do ciclo estral. A integridade da MP não foi afetada (P 0,05) pela incubação com FO e CEO na fase folicular ou luteal. Incubação com FO independente do estágio do ciclo estral apresentou maior (P < 0,05) taxa de RA em comparação ao grupo controle negativo após longo tempo de incubação (18-24 h) enquanto que a interação das CEO (folicular ou luteal) reduziu (P < 0,05) a taxa de capacitação espermática em comparação ao grupo controle positivo ao longo da incubação. Conclui-se que utilizando a espécie ovina como modelo, a adição do FO na fase folicular ou luteal promove um aumento na cinemática espermática até 4 h de incubação e na taxa de RA após 18-24 h, enquanto que a interação das CEO com SPZ reduz a cinemática espermática e atrasa a capacitação espermática in vitro.

Oviduct fluid (OF) provides a suitable environment for the sperm capacitation and fertilization. Still, oviductal epithelial cell (OEC) interacts with sperm prolonging sperm lifespan. Evidences on literature suggest that OF varies according to the stage of estrous cycle, however, there are only few reports about the effect of this variation on sperm function. Thus, the objective of the study was to evaluate the effect of OF at follicular and luteal phases and the interaction of sperm with OEC on the modulation of sperm function and capacitation/acrosome react (AR) processes. To achieve this goal, two experiments were performed. For this, OF at follicular and luteal phases (classification based on ovarian morphology) and OEC were obtained from cow oviducts at a slaughterhouse. For both experiments, after swim-up technique (pool of three rams) sperm were randomly distributed in the experimental groups. In Experiment 1, 8 x 106 sperm/mL were added in the medium: i) Fert-TALP (positive control), ii) Fert-TALP without capacitating substance (modified Fert-TALP; negative control), iii) modified Fert-TALP supplemented with 10% of OF at follicular phase or iv) modified Fert-TALP supplemented with 10% of OF at luteal phase. In Experiment 2, to mimic oviductal conditions during the follicular and luteal phases, the OECs were cultured in TCM-199 medium supplemented with 10% of fetal bovine serum until confluence. Previously (24 h) to co-culture with sperm, the follicular and luteal conditions were induced by supplementation of 17beta-estradiol (E2) and progesterone (P4) hormones in the Fert-TALP medium, at concentrations detected in the oviduct during these phases. For co-culture, 8 x 106 sperm/mL were added in the presence of OEC monolayer: i) OEC follicular phase co-culture of sperm and OEC in Fert-TALP medium supplemented with 290 pg/mL E2 and 6 ng/mL P4 (concentration similar to follicular phase); ii) OEC luteal phase co-culture of sperm and OEC in FertTALP medium supplemented with 80 pg/mL E2 and 85 ng/mL P4 (concentration similar to luteal phase); iii) sperm culture in Fert-TALP medium supplemented with 290 pg/mL E2 and 6 ng/mL P4 and iv) sperm culture in Fert-TALP medium supplemented with 80 pg/mL E2 and 85 ng/mL P4. The positive control was the conventional Fert-TALP medium. Sperm were incubated at 38 ºC in 5% CO2 and the parameters of sperm kinematics, plasma membrane (PM) integrity and sperm capacitation status were evaluated after 0, 2, 4, 6, 18 and 24 h. The variables were subjected to a repeated measure two-way ANOVA (Bonferroni; P < 0.05). The results of the Experiment 1 showed that incubation up to four hours with OF regardless the stage of estrous cycle promoted an increase in most of kinematic parameters similar (P 0.05) to the positive control group while in Experiment 2, co-culture between sperm and OEC, regardless the stage of estrous cycle, reduced (P < 0.05) these kinematic parameters. The PM integrity was not affected (P 0.05) by incubation with OF and OEC at follicular or luteal phase. Incubation with OF regardless the phase of estrous cycle presented higher (P < 0.05) AR rate compared to the negative control group after a long-time incubation (18-24 h), while the interaction of OEC (either follicular or luteal stage) reduced (P < 0.05) sperm capacitation rate compare to positive control group throughout incubation. It is concluded that the supplementation with OF either at follicular or luteal stage into the medium, promotes an increase in the sperm kinematic for up to 4 h of incubation and AR rate after 18-24 h. In contrast, interaction between sperm and OEC reduce sperm kinematics and delay sperm capacitation in vitro using the ovine specie as a model.