RESUMO

The objectives of this study were to evaluate goat sperm sorting in continuous Percoll® density gradients and gamete freezability, in the presence or absence of phenolic antioxidants. For this, semen pools were sorted, frozen, and evaluated. The non-selected group (NSg) presented lower progressive motility (PM), linearity (LIN), straightness (STR), and wobble (WOB) than the selected groups, and straight line velocity (VSL) compared to those with catechin or resveratrol. The amplitude of lateral head displacement (ALH) was higher in NSg, and quercetin reduced the mitochondrial membrane potential (MMP). After thawing, the NSg presented lower PM than the selected groups, VSL and VAP (average path velocity) than the selected group with or without catechin, LIN and WOB than the selected with or without catechin or resveratrol, and STR than the selected with catechin. Moreover, NSg presented higher ALH and BCF than the samples selected with or without catechin. Plasma membrane integrity and intact and living cells were higher in the selected groups, and MMP was lower in the NSg and the selected group with quercetin. Thus, centrifugation in Percoll® continuous density gradients is a viable methodology to select goat sperm compatible with the freezing, especially in the presence of catechin or resveratrol.(AU)

Objetivou-se avaliar a separação de espermatozoides caprinos em gradientes de densidade contínuos de Percoll® e a congelabilidade espermática, com ou sem antioxidantes fenólicos. Para tal, pools seminais foram selecionados, congelados e avaliados. O grupo não selecionado (gNS) apresentou menor motilidade progressiva (MP), linearidade (LIN), retilinearidade (STR) e oscilação (WOB) do que os selecionados, bem como menor velocidade linear progressiva (VSL) do que os com catequina ou resveratrol. A amplitude de deslocamento lateral de cabeça (ALH) foi maior no gNS e a quercetina reduziu o potencial de membrana mitocondrial (PMM). Após a descongelação, o gNS manifestou menor MP do que os selecionados, menor VSL e VAP (velocidade média da trajetória) do que os com ou sem catequina, menor LIN e WOB do que os com ou sem catequina ou resveratrol, e menor STR do que os com catequina, além de maior ALH e BCF do que os com ou sem catequina. A integridade da membrana plasmática e as células intactas e vivas foram maiores nas amostras selecionadas e o PMM foi inferior no gNS e no selecionado com quercetina. Portanto, a centrifugação em gradientes contínuos de densidade de Percoll® é uma metodologia viável para selecionar espermatozoides caprinos compatíveis com a congelação, especialmente na presença de catequina ou resveratrol.(AU)

Assuntos

RESUMO

The objectives of this study were to evaluate goat sperm sorting in continuous Percoll® density gradients and gamete freezability, in the presence or absence of phenolic antioxidants. For this, semen pools were sorted, frozen, and evaluated. The non-selected group (NSg) presented lower progressive motility (PM), linearity (LIN), straightness (STR), and wobble (WOB) than the selected groups, and straight line velocity (VSL) compared to those with catechin or resveratrol. The amplitude of lateral head displacement (ALH) was higher in NSg, and quercetin reduced the mitochondrial membrane potential (MMP). After thawing, the NSg presented lower PM than the selected groups, VSL and VAP (average path velocity) than the selected group with or without catechin, LIN and WOB than the selected with or without catechin or resveratrol, and STR than the selected with catechin. Moreover, NSg presented higher ALH and BCF than the samples selected with or without catechin. Plasma membrane integrity and intact and living cells were higher in the selected groups, and MMP was lower in the NSg and the selected group with quercetin. Thus, centrifugation in Percoll® continuous density gradients is a viable methodology to select goat sperm compatible with the freezing, especially in the presence of catechin or resveratrol.(AU)

Objetivou-se avaliar a separação de espermatozoides caprinos em gradientes de densidade contínuos de Percoll® e a congelabilidade espermática, com ou sem antioxidantes fenólicos. Para tal, pools seminais foram selecionados, congelados e avaliados. O grupo não selecionado (gNS) apresentou menor motilidade progressiva (MP), linearidade (LIN), retilinearidade (STR) e oscilação (WOB) do que os selecionados, bem como menor velocidade linear progressiva (VSL) do que os com catequina ou resveratrol. A amplitude de deslocamento lateral de cabeça (ALH) foi maior no gNS e a quercetina reduziu o potencial de membrana mitocondrial (PMM). Após a descongelação, o gNS manifestou menor MP do que os selecionados, menor VSL e VAP (velocidade média da trajetória) do que os com ou sem catequina, menor LIN e WOB do que os com ou sem catequina ou resveratrol, e menor STR do que os com catequina, além de maior ALH e BCF do que os com ou sem catequina. A integridade da membrana plasmática e as células intactas e vivas foram maiores nas amostras selecionadas e o PMM foi inferior no gNS e no selecionado com quercetina. Portanto, a centrifugação em gradientes contínuos de densidade de Percoll® é uma metodologia viável para selecionar espermatozoides caprinos compatíveis com a congelação, especialmente na presença de catequina ou resveratrol.(AU)

Assuntos

RESUMO

The aim of this study was to evaluate the effects of different concentrations of (+)-catechin or (-)-epigallocatechin gallate (EGCG) on goat semen freezability. Poolsof semen were processed (Experiment 1: 0, 15, 25, 50, 75, or 100µM (+)-catechin; Experiment 2: 0, 15, 25, 50, 75, or 100µM EGCG) and frozen. After thawing, the samples were evaluated for kinematics, plasma membrane (PMi) and acrosome integrity, morphology, and oxidative stress, at 0 and 1h. In Experiment 1, at 0h, VSL and VAP were greater (P<0.05) with 15µM than with 50 and 100; WOB was lower (P<0.05) with 100µM than with 0, 15, and 25; and BCF was higher (P<0.05) with 75 and 100µM than with 0. In turn, in Experiment 2, progressive motility was higher (P<0.05) with0 and 15µM than with50 and 75; LIN was lower (P<0.05) with75 and100µM than with0 and 15; WOB was higher (P<0.05) with0 and 15µM; and PMi was greater (P<0.05) with100µM than 0. Thus, (+)-catechin or EGCG at higher concentrations inhibits the kinematics of frozen goat sperm, in a transitory way, and 100µM of EGCG preserves the PMi.(AU)

Objetivou-se avaliar o efeito de diferentes concentrações de (+)-catequina ou (-)-epigalocatequina galato (EGCG) sobre a congelabilidade do sêmen caprino. Poolsseminais foram processados (experimento 1: 0, 15, 25, 50, 75 ou 100µM de (+)-catequina; experimento 2: 0, 15, 25, 50, 75 ou 100µM de EGCG) e congelados. Após a descongelação, foram avaliadas a cinética, a integridade de membrana plasmática (iMP) e acrossomal, a morfologia e o estresse oxidativo, a zero e a uma hora. No experimento 1, a zero hora, VSL e VAP foram maiores (P<0,05) com 15µM do que com 50 e100; WOB foi menor (P<0,05) com 100µM do que com 0, 15 e 25; e BCF foi maior (P<0,05) com 75 e 100µM do que com 0. No experimento 2, a motilidade progressiva foi maior (P<0,05) com 0 e 15µM do que com 50 e 75; LIN foi menor (P<0,05) com 75 e 100µM do que com 0 e 15; WOB foi maior (P<0,05) com 0 e 15µM; e iMP foi maior (P<0,05) com 100µM do que com 0. Assim, (+)-catequina ou EGCG em altas concentrações inibem, transitoriamente, a cinética de espermatozoides congelados caprinos, e 100µM de EGCG preserva a iMP.(AU)

Assuntos

RESUMO

The aim of this study was to evaluate the effects of different concentrations of (+)-catechin or (-)-epigallocatechin gallate (EGCG) on goat semen freezability. Poolsof semen were processed (Experiment 1: 0, 15, 25, 50, 75, or 100µM (+)-catechin; Experiment 2: 0, 15, 25, 50, 75, or 100µM EGCG) and frozen. After thawing, the samples were evaluated for kinematics, plasma membrane (PMi) and acrosome integrity, morphology, and oxidative stress, at 0 and 1h. In Experiment 1, at 0h, VSL and VAP were greater (P<0.05) with 15µM than with 50 and 100; WOB was lower (P<0.05) with 100µM than with 0, 15, and 25; and BCF was higher (P<0.05) with 75 and 100µM than with 0. In turn, in Experiment 2, progressive motility was higher (P<0.05) with0 and 15µM than with50 and 75; LIN was lower (P<0.05) with75 and100µM than with0 and 15; WOB was higher (P<0.05) with0 and 15µM; and PMi was greater (P<0.05) with100µM than 0. Thus, (+)-catechin or EGCG at higher concentrations inhibits the kinematics of frozen goat sperm, in a transitory way, and 100µM of EGCG preserves the PMi.(AU)

Objetivou-se avaliar o efeito de diferentes concentrações de (+)-catequina ou (-)-epigalocatequina galato (EGCG) sobre a congelabilidade do sêmen caprino. Poolsseminais foram processados (experimento 1: 0, 15, 25, 50, 75 ou 100µM de (+)-catequina; experimento 2: 0, 15, 25, 50, 75 ou 100µM de EGCG) e congelados. Após a descongelação, foram avaliadas a cinética, a integridade de membrana plasmática (iMP) e acrossomal, a morfologia e o estresse oxidativo, a zero e a uma hora. No experimento 1, a zero hora, VSL e VAP foram maiores (P<0,05) com 15µM do que com 50 e100; WOB foi menor (P<0,05) com 100µM do que com 0, 15 e 25; e BCF foi maior (P<0,05) com 75 e 100µM do que com 0. No experimento 2, a motilidade progressiva foi maior (P<0,05) com 0 e 15µM do que com 50 e 75; LIN foi menor (P<0,05) com 75 e 100µM do que com 0 e 15; WOB foi maior (P<0,05) com 0 e 15µM; e iMP foi maior (P<0,05) com 100µM do que com 0. Assim, (+)-catequina ou EGCG em altas concentrações inibem, transitoriamente, a cinética de espermatozoides congelados caprinos, e 100µM de EGCG preserva a iMP.(AU)

Assuntos

RESUMO

O objetivo deste estudo foi avaliar o efeito da suplementação do diluidor de congelação de sêmen ovino com o flavonoide miricetina contra os danos ocasionados aos espermatozoides. Oito pools de sêmen, obtidos de quatro reprodutores ovinos, foram congelados com diferentes concentrações de miricetina (0, 1, 10, 100 e 1000nM). Após o descongelamento, o sêmen foi avaliado quanto à cinética espermática, à integridade das membranas plasmática e acrossomal, ao potencial de membrana mitocondrial, aos níveis de ROS intracelular, à peroxidação lipídica e à estabilidade de membrana. Amostras tratadas com miricetina 10nM apresentaram menor percentual de células rápidas (P≤0,05), quando comparadas ao grupo miricetina 1000nM. Amostras do grupo controle apresentaram maior (P≤0,05) VAP que o grupo 10nM de miricetina, enquanto amostras criopreservadas com miricetina (10, 100 e 1000nM) evidenciaram maior (P<0,05) BCF, quando comparadas ao grupo controle. O grupo tratado com miricetina 1000nM apresentou maior percentual (P<0,05) de células com peroxidação lipídica, quando comparado ao grupo controle. Em conclusão, a suplementação do diluidor de criopreservação de sêmen ovino com 10 e 100nM de miricetina afeta a cinética espermática sem provocar alterações na estrutura geral do gameta, enquanto 1000nM de miricetina provoca mudanças na cinética associadas à danos peroxidativos.(AU)

The aim of this study was to evaluate the effect of the supplementation of ram semen frozen with extender with the flavonoid myricetin against damage to sperm. Eight pools of semen obtained from four ram breeders, were frozen with different concentrations of myicetin (0, 1, 10, 100 and 1000nM). After thawing, the semen was evaluated for spermatic kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, intracellular ROS levels, lipid peroxidation, and membrane stability. Samples treated with 10nM myricetin preserved a lower percentage of rapid cells (P≤0.05) when compared to the 1000nM myricetin group. Samples from the control group presented higher (P≤0.05) VAP than 10nM group of myricetin, while cryopreserved samples with myicetin (10, 100 and 1000nM) showed greater (P<0.05) BCF, when compared to control group. The group treated with 1000nM myricetin had a higher percentage (P<0.05) of cells with lipid peroxidation, when compared to the control group. In conclusion, supplementation of ram semen cryopreservation extender with 10 and 100nM myricetin affects sperm kinetics, without causing changes in the overall structure of the gamete, while 1000nM myricetin causes changes in the kinetics associated with peroxidative damage.(AU)

Assuntos

RESUMO

O objetivo deste estudo foi avaliar o efeito da suplementação do diluidor de congelação de sêmen ovino com o flavonoide miricetina contra os danos ocasionados aos espermatozoides. Oito pools de sêmen, obtidos de quatro reprodutores ovinos, foram congelados com diferentes concentrações de miricetina (0, 1, 10, 100 e 1000nM). Após o descongelamento, o sêmen foi avaliado quanto à cinética espermática, à integridade das membranas plasmática e acrossomal, ao potencial de membrana mitocondrial, aos níveis de ROS intracelular, à peroxidação lipídica e à estabilidade de membrana. Amostras tratadas com miricetina 10nM apresentaram menor percentual de células rápidas (P≤0,05), quando comparadas ao grupo miricetina 1000nM. Amostras do grupo controle apresentaram maior (P≤0,05) VAP que o grupo 10nM de miricetina, enquanto amostras criopreservadas com miricetina (10, 100 e 1000nM) evidenciaram maior (P<0,05) BCF, quando comparadas ao grupo controle. O grupo tratado com miricetina 1000nM apresentou maior percentual (P<0,05) de células com peroxidação lipídica, quando comparado ao grupo controle. Em conclusão, a suplementação do diluidor de criopreservação de sêmen ovino com 10 e 100nM de miricetina afeta a cinética espermática sem provocar alterações na estrutura geral do gameta, enquanto 1000nM de miricetina provoca mudanças na cinética associadas à danos peroxidativos.(AU)

The aim of this study was to evaluate the effect of the supplementation of ram semen frozen with extender with the flavonoid myricetin against damage to sperm. Eight pools of semen obtained from four ram breeders, were frozen with different concentrations of myicetin (0, 1, 10, 100 and 1000nM). After thawing, the semen was evaluated for spermatic kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, intracellular ROS levels, lipid peroxidation, and membrane stability. Samples treated with 10nM myricetin preserved a lower percentage of rapid cells (P≤0.05) when compared to the 1000nM myricetin group. Samples from the control group presented higher (P≤0.05) VAP than 10nM group of myricetin, while cryopreserved samples with myicetin (10, 100 and 1000nM) showed greater (P<0.05) BCF, when compared to control group. The group treated with 1000nM myricetin had a higher percentage (P<0.05) of cells with lipid peroxidation, when compared to the control group. In conclusion, supplementation of ram semen cryopreservation extender with 10 and 100nM myricetin affects sperm kinetics, without causing changes in the overall structure of the gamete, while 1000nM myricetin causes changes in the kinetics associated with peroxidative damage.(AU)

Assuntos

RESUMO

This work aimed to evaluate the effect of stabilisation times, glycerol concentration, and the catalase and superoxide dismutase supplementation of diluent on parameters of frozen-thawed spermatozoa from epididymis of Nelore bulls: Experiment 1: spermatozoa diluted in Tris-egg yolk with glycerol (3%, 5% or 7%) and stabilisation times (0, 2 or 4 hr at 5°C); Experiment 2: Tris-egg yolk only, Tris-egg yolk with catalase (CAT, 50 or 100 U ml-1 ) or superoxide dismutase (SOD, 50 or 100 U ml-1 ). Frozen-thawed spermatozoa were evaluated for kinetic parameters, plasma membrane and acrosome integrity, mitochondrial activity and IVF capacity. ALH and BCF were affected (p < .05) by glycerol at 3% after 4-hr equilibration time and 7% after 2-hr equilibration time. Glycerol 3% had lower (p < .05) iPM and iAc after 4 hr. Glycerol 5% had greater (p < .05) hPMM after 4 hr and iAc after 2 hr than at 0 hr. SOD 100 U ml-1 had lower (p < .05) linearity and wobble compared to control group. No was observed differences to fertilisation rate (p < .05) among groups. In conclusion, glycerol 5% in Tris-egg yolk extender for 4 hr is suitable for the preservation of sperm kinetics and membrane integrity. CAT (50 and 100 U ml-1 ) or SOD (50-100 U ml-1 ) had no beneficial effects on sperm kinetics, plasma and acrosomal membrane integrity, mitochondrial activity or the capacity for IVF of frozen-thawed spermatozoa from epididymis of Nelore bulls.

Assuntos

RESUMO

The aim of this study was to evaluate the effect of different concentrations of trans-resveratrol or quercetin on the ability of goat sperm to withstand being frozen. Six pools of semen obtained from six male goats were processed with different concentrations of resveratrol or quercetin (Experiment 1: 0, 15, 25, 50, 75 or 100µM resveratrol; Experiment 2: 0, 15, 25, 50, 75 or 100µM quercetin) and frozen. After thawing, the semen was evaluated for sperm kinematics, plasma membrane and acrosome integrity, morphology and oxidative stress following 0 and 1h of incubation. Immediately after thawing (0h), wobble (oscillation index) in the groups treated with 100µM of quercetin or resveratrol was lower (P<0.05) than in those treated with 0 and 25µM resveratrol and 0µM quercetin, respectively. After 1h of incubation, the total motility in treatments with 15, 50 and 75µM quercetin, as well as the plasma membrane integrity in all quercetin concentrations were lower (P<0.05) than at 0h. In opposition, the linearity of semen samples treated with 100µM quercetin and the straightness of those treated with 75 and 100µM quercetin were lower (P<0.05) at 0h than at 1h after thawing. Thus, it can be concluded that resveratrol and quercetin at high concentrations (100µM) transiently reduce the wobble of goat sperm submitted to frozen storage, and that quercetin (75 and 100µM) increases the linearity and straightness over time, which can be favorable for fertility.(AU)

O objetivo deste estudo foi avaliar o efeito de diferentes concentrações de transresveratrol ou quercetina sobre a capacidade dos espermatozoides caprinos de resistirem à congelação. Seis pools de sêmen, obtidos de seis reprodutores caprinos, foram processados com diferentes concentrações de resveratrol ou quercetina (Experimento 1: 0, 15, 25, 50, 75 ou 100µM de resveratrol; Experimento 2: 0, 15, 25, 50, 75 ou 100µM de quercetina) e congelados. Após o descongelamento, o sêmen foi avaliado quanto à cinética espermática, à integridade das membranas plasmática e acrossomal, à morfologia e ao estresse oxidativo nos tempos zero e uma hora de incubação. Imediatamente após a descongelação (zero hora), o wobble (índice de oscilação) nos grupos tratados com 100µM de quercetina ou de resveratrol foi menor (P<0,05) do que nos tratados com 0 e 25µM de resveratrol e com 0µM de quercetina, respectivamente. Após uma hora de incubação, a motilidade total dos tratamentos com 15, 50 e 75µM de quercetina, assim como a integridade de membrana plasmática em todas as concentrações de quercetina, foi menor (P<0,05) do que à zero hora. Em oposição, a linearidade das amostras de sêmen tratadas com 100µM de quercetina e a retilinearidade daquelas tratadas com 75µM e 100µM de quercetina foram menores (P<0,05) à zero hora do que à uma hora após descongelação. Assim, pode-se concluir que o resveratrol e a quercetina, em concentrações elevadas (100µM), reduzem, transitoriamente, o índice de oscilação de espermatozoides caprinos submetidos à congelação e que a quercetina (75 e 100µM) aumenta a linearidade e a retilinearidade ao longo do tempo, o que pode ser favorável à fertilidade.(AU)

Assuntos

RESUMO

The aim of this study was to evaluate the effect of different concentrations of trans-resveratrol or quercetin on the ability of goat sperm to withstand being frozen. Six pools of semen obtained from six male goats were processed with different concentrations of resveratrol or quercetin (Experiment 1: 0, 15, 25, 50, 75 or 100µM resveratrol; Experiment 2: 0, 15, 25, 50, 75 or 100µM quercetin) and frozen. After thawing, the semen was evaluated for sperm kinematics, plasma membrane and acrosome integrity, morphology and oxidative stress following 0 and 1h of incubation. Immediately after thawing (0h), wobble (oscillation index) in the groups treated with 100µM of quercetin or resveratrol was lower (P<0.05) than in those treated with 0 and 25µM resveratrol and 0µM quercetin, respectively. After 1h of incubation, the total motility in treatments with 15, 50 and 75µM quercetin, as well as the plasma membrane integrity in all quercetin concentrations were lower (P<0.05) than at 0h. In opposition, the linearity of semen samples treated with 100µM quercetin and the straightness of those treated with 75 and 100µM quercetin were lower (P<0.05) at 0h than at 1h after thawing. Thus, it can be concluded that resveratrol and quercetin at high concentrations (100µM) transiently reduce the wobble of goat sperm submitted to frozen storage, and that quercetin (75 and 100µM) increases the linearity and straightness over time, which can be favorable for fertility.(AU)

O objetivo deste estudo foi avaliar o efeito de diferentes concentrações de transresveratrol ou quercetina sobre a capacidade dos espermatozoides caprinos de resistirem à congelação. Seis pools de sêmen, obtidos de seis reprodutores caprinos, foram processados com diferentes concentrações de resveratrol ou quercetina (Experimento 1: 0, 15, 25, 50, 75 ou 100µM de resveratrol; Experimento 2: 0, 15, 25, 50, 75 ou 100µM de quercetina) e congelados. Após o descongelamento, o sêmen foi avaliado quanto à cinética espermática, à integridade das membranas plasmática e acrossomal, à morfologia e ao estresse oxidativo nos tempos zero e uma hora de incubação. Imediatamente após a descongelação (zero hora), o wobble (índice de oscilação) nos grupos tratados com 100µM de quercetina ou de resveratrol foi menor (P<0,05) do que nos tratados com 0 e 25µM de resveratrol e com 0µM de quercetina, respectivamente. Após uma hora de incubação, a motilidade total dos tratamentos com 15, 50 e 75µM de quercetina, assim como a integridade de membrana plasmática em todas as concentrações de quercetina, foi menor (P<0,05) do que à zero hora. Em oposição, a linearidade das amostras de sêmen tratadas com 100µM de quercetina e a retilinearidade daquelas tratadas com 75µM e 100µM de quercetina foram menores (P<0,05) à zero hora do que à uma hora após descongelação. Assim, pode-se concluir que o resveratrol e a quercetina, em concentrações elevadas (100µM), reduzem, transitoriamente, o índice de oscilação de espermatozoides caprinos submetidos à congelação e que a quercetina (75 e 100µM) aumenta a linearidade e a retilinearidade ao longo do tempo, o que pode ser favorável à fertilidade.(AU)

Assuntos

RESUMO

The aim of this study was to evaluate the fertilization rate of cows that were superovulated and artificially inseminated with sex-sorted semen. Cows were treated with an intravaginal progesterone device plus estradiol benzoate (day 0). Superstimulation treatments began four days after with eight applications of FSH at 12 h intervals. D-Cloprostenol was administered on day 6. Progesterone device was removed on day 7, and LH was administered on day 8. The treatments were divided as follows: NonSx, two AI with non-sorted semen were conducted 12 and 24 h after LH; Sx12&24, two AI with sex-sorted semen were conducted 12 and 24 h after LH; and Sx24&36, two AI with sex-sorted semen were conducted 24 and 36 h after LH. Embryos were recovered on day 16 and were evaluated and classified. Percentage of fertilized embryos tended to be greater for the non-sorted semen than the sex-sorted semen. The number of unfertilized oocytes was smaller when the non-sorted semen was used relative to the sex-sorted semen. There was no difference between the treatments that used sexed semen. In conclusion, the use of sex-sorted semen in superovulated dairy cows results in greater numbers of unfertilized oocytes than non-sorted. However, when only sorted semen is used AI should be performed 24 and 36 h after LH.(AU)

Assuntos

RESUMO

The aim of this study was to evaluate the fertilization rate of cows that were superovulated and artificially inseminated with sex-sorted semen. Cows were treated with an intravaginal progesterone device plus estradiol benzoate (day 0). Superstimulation treatments began four days after with eight applications of FSH at 12 h intervals. D-Cloprostenol was administered on day 6. Progesterone device was removed on day 7, and LH was administered on day 8. The treatments were divided as follows: NonSx, two AI with non-sorted semen were conducted 12 and 24 h after LH; Sx12&24, two AI with sex-sorted semen were conducted 12 and 24 h after LH; and Sx24&36, two AI with sex-sorted semen were conducted 24 and 36 h after LH. Embryos were recovered on day 16 and were evaluated and classified. Percentage of fertilized embryos tended to be greater for the non-sorted semen than the sex-sorted semen. The number of unfertilized oocytes was smaller when the non-sorted semen was used relative to the sex-sorted semen. There was no difference between the treatments that used sexed semen. In conclusion, the use of sex-sorted semen in superovulated dairy cows results in greater numbers of unfertilized oocytes than non-sorted. However, when only sorted semen is used AI should be performed 24 and 36 h after LH.

Assuntos

RESUMO

The aim of this study was to investigate the effects of catalase and pre-freezing equilibration during ram sperm cryopreservation on motility and membrane and acrosomal integrity of frozen-thawed semen, as well as conception rate following laparoscopic timedinsemination. Semen was collected from four mature Dorper rams, pooled and diluted in Tris egg-yolk extender basic solution (CON), or this solution supplemented with catalase (CAT; 20 U/100 × 106 sperm). Extended semen was packaged in 0.25 ml mini straws (25 × 106 sperm/straw), chilled (to 5°C), and then either frozen immediately (CON and CAT) or maintained at 5°C for 12 h of pre-freezing equilibration (CON12 and CAT12). Immediately after thawing and at 1 h after incubation at 37°C, kinematic parameters (CASA), plasma membrane integrity (PI-FITC), and acrosomal status (FITC-PNA) of sperm were assessed. There were no significant differences among the four groups on sperm traits evaluated immediately postthaw. However, after 1 h of incubation, total motility (46.7 and 25.0%) and plasma membrane integrity (38.7 and 25.7%) were higher (P < 0.05) in CAT12 than CON. When these two treatments were used for laparoscopic timed artificial insemination of ewes (with synchronized ovulation), conception rate was similar for CAT12 and CON (32.8%, n = 61 vs. 27.3%, n = 55). In conclusion, the combination of catalase and pre-freezing equilibration resulted in significantly improved quality of post-thawed ram semen without affecting conception rate in fixed-time laparoscopically intrauterine inseminated ewes.(AU)

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RESUMO

The aim of this study was to investigate the effects of catalase and pre-freezing equilibration during ram sperm cryopreservation on motility and membrane and acrosomal integrity of frozen-thawed semen, as well as conception rate following laparoscopic timedinsemination. Semen was collected from four mature Dorper rams, pooled and diluted in Tris egg-yolk extender basic solution (CON), or this solution supplemented with catalase (CAT; 20 U/100 × 106 sperm). Extended semen was packaged in 0.25 ml mini straws (25 × 106 sperm/straw), chilled (to 5°C), and then either frozen immediately (CON and CAT) or maintained at 5°C for 12 h of pre-freezing equilibration (CON12 and CAT12). Immediately after thawing and at 1 h after incubation at 37°C, kinematic parameters (CASA), plasma membrane integrity (PI-FITC), and acrosomal status (FITC-PNA) of sperm were assessed. There were no significant differences among the four groups on sperm traits evaluated immediately postthaw. However, after 1 h of incubation, total motility (46.7 and 25.0%) and plasma membrane integrity (38.7 and 25.7%) were higher (P < 0.05) in CAT12 than CON. When these two treatments were used for laparoscopic timed artificial insemination of ewes (with synchronized ovulation), conception rate was similar for CAT12 and CON (32.8%, n = 61 vs. 27.3%, n = 55). In conclusion, the combination of catalase and pre-freezing equilibration resulted in significantly improved quality of post-thawed ram semen without affecting conception rate in fixed-time laparoscopically intrauterine inseminated ewes.

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O objetivo deste trabalho foi comparar o resultado de diferentes protocolos de inseminação artificial emtempo fixo (IATF), utilizando-se sêmen sexado, sobre a eficiência reprodutiva em novilhas Girolando. Foramutilizadas 62 novilhas clinicamente hígidas e com escore corporal entre 2,5 e 3,5, divididas aleatoriamente emtrês grupos: controle (n = 21), FSH/LH (n = 21) e eCG (n = 20). Todos os animais receberam o mesmotratamento hormonal para sincronização do estro, consistindo na introdução de dispositivo intravaginal com750mg de progesterona (P4) no dia zero às 17-h, e aplicação de 2-mg de benzoato de estradiol (BE). No dia oitoàs 17-h, foram retirados os implantes e aplicados ml de prostaglandina (0,500-mg cloprostenol). No dia nove,todos os animais receberam 1-mg de benzoato de estradiol (BE) às 17-h. As novilhas foram inseminadas no dia11, às cinco horas, isto é 60-h após a retirada do implante. No dia oito, as novilhas foram distribuídasaleatoriamente em três grupos: grupo controle sem FSH/LH e eCG: grupo FSH/LH 25-UI de FSH e LHaplicados na retirada do implante: grupo eCG 300-UI de eCG aplicados na retirada do implante. As novilhasforam examinadas por ultrassonografia 35 dias após a IA para diagnóstico de gestação e aos 45 dias paraavaliação de perda embrionária. O percentual de prenhez para os grupos controle, FSH/LH e eCG foramrespectivamente 19, 28 e 35%. Os dados foram avaliados pelo teste do qui-quadrado, com nível de significânciade 5%. Não houve diferença significativa entre os grupos. Ao se avaliar a taxa de prenhez entre o grupo controlee o grupo eCG em novilhas que não estavam ciclando, houve uma diferença significativa. Conclui-se que, emanimais cíclicos, a eCG e o FSH/LH não interferiram nas taxas de prenhez. Entretanto, quando comparados comos animais acíclicos ou que tinham folículos menores que 8-mm, a eCG teve um efeito favorável, melhorandosubstancialmente os resultados.

The aim of this study was to compare results of different protocols of timed artificial insemination (TAI)using sexed semen on reproductive efficiency in Girolando cattle. Sixty two heifers were used, being clinicallyhealthy with a body score condition between 2,5 and 3,5, divided randomly into three groups: control (n = 21),FSH/LH (n = 21) and eCG (n = 20). All animals received the same hormonal treatment to synchronize estrus,consisting in inserting na intravaginal device with 750 mg of progesterone (P4) plus 2 mg of estradiol benzoate(EB) on day 0 (5:00 PM). On day 8 (5:00 PM), the device was removed, and 2 ml of prostaglandin (0,5 mg ofcloprostenol) were administered. On day 9 all animals received 1 mg of estradiol benzoate (EB; 5:00 PM), andwere inseminated on day 11 at 5:00 AM, 60 h after device removal. On day 8, heifers were randomly assigned intothree groups. Control group without FSH/LH and eCG; group FSH/LH - 25 IU FSH and LH administered afterdevice removal, group eCG - 300 IU eCG administered after device removal. Heifers were examined byultrasonography 35 days after AI for pregnancy diagnosis and 45 days for evaluation of embryonic death.Pregnancy rates for control, FSH/LH, and eCG groups were, respectively, 19, 28 and 35%. Data were analyzed bychi-square, with 5% significance level. No significant difference was seen between-groups. When control and eCGgroups were, compared in heifers not cycling, a difference in pregnancy rate was observed. It is concluded that incycling animals, eCG and FSH/LH does not interfere with pregnancy rates, however in those heifers that were notcycling or had follicles smaller than 8 mm, eCG had a favorable effect thereby increasing substantially the results.

Assuntos

RESUMO

O objetivo deste trabalho foi comparar o resultado de diferentes protocolos de inseminação artificial emtempo fixo (IATF), utilizando-se sêmen sexado, sobre a eficiência reprodutiva em novilhas Girolando. Foramutilizadas 62 novilhas clinicamente hígidas e com escore corporal entre 2,5 e 3,5, divididas aleatoriamente emtrês grupos: controle (n = 21), FSH/LH (n = 21) e eCG (n = 20). Todos os animais receberam o mesmotratamento hormonal para sincronização do estro, consistindo na introdução de dispositivo intravaginal com750mg de progesterona (P4) no dia zero às 17-h, e aplicação de 2-mg de benzoato de estradiol (BE). No dia oitoàs 17-h, foram retirados os implantes e aplicados ml de prostaglandina (0,500-mg cloprostenol). No dia nove,todos os animais receberam 1-mg de benzoato de estradiol (BE) às 17-h. As novilhas foram inseminadas no dia11, às cinco horas, isto é 60-h após a retirada do implante. No dia oito, as novilhas foram distribuídasaleatoriamente em três grupos: grupo controle sem FSH/LH e eCG: grupo FSH/LH 25-UI de FSH e LHaplicados na retirada do implante: grupo eCG 300-UI de eCG aplicados na retirada do implante. As novilhasforam examinadas por ultrassonografia 35 dias após a IA para diagnóstico de gestação e aos 45 dias paraavaliação de perda embrionária. O percentual de prenhez para os grupos controle, FSH/LH e eCG foramrespectivamente 19, 28 e 35%. Os dados foram avaliados pelo teste do qui-quadrado, com nível de significânciade 5%. Não houve diferença significativa entre os grupos. Ao se avaliar a taxa de prenhez entre o grupo controlee o grupo eCG em novilhas que não estavam ciclando, houve uma diferença significativa. Conclui-se que, emanimais cíclicos, a eCG e o FSH/LH não interferiram nas taxas de prenhez. Entretanto, quando comparados comos animais acíclicos ou que tinham folículos menores que 8-mm, a eCG teve um efeito favorável, melhorandosubstancialmente os resultados.(AU)

The aim of this study was to compare results of different protocols of timed artificial insemination (TAI)using sexed semen on reproductive efficiency in Girolando cattle. Sixty two heifers were used, being clinicallyhealthy with a body score condition between 2,5 and 3,5, divided randomly into three groups: control (n = 21),FSH/LH (n = 21) and eCG (n = 20). All animals received the same hormonal treatment to synchronize estrus,consisting in inserting na intravaginal device with 750 mg of progesterone (P4) plus 2 mg of estradiol benzoate(EB) on day 0 (5:00 PM). On day 8 (5:00 PM), the device was removed, and 2 ml of prostaglandin (0,5 mg ofcloprostenol) were administered. On day 9 all animals received 1 mg of estradiol benzoate (EB; 5:00 PM), andwere inseminated on day 11 at 5:00 AM, 60 h after device removal. On day 8, heifers were randomly assigned intothree groups. Control group without FSH/LH and eCG; group FSH/LH - 25 IU FSH and LH administered afterdevice removal, group eCG - 300 IU eCG administered after device removal. Heifers were examined byultrasonography 35 days after AI for pregnancy diagnosis and 45 days for evaluation of embryonic death.Pregnancy rates for control, FSH/LH, and eCG groups were, respectively, 19, 28 and 35%. Data were analyzed bychi-square, with 5% significance level. No significant difference was seen between-groups. When control and eCGgroups were, compared in heifers not cycling, a difference in pregnancy rate was observed. It is concluded that incycling animals, eCG and FSH/LH does not interfere with pregnancy rates, however in those heifers that were notcycling or had follicles smaller than 8 mm, eCG had a favorable effect thereby increasing substantially the results.(AU)

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RESUMO

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml(-1) ] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post-thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml(-1) Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml(-1) groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml(-1) ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.

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Successful DNA extraction is indispensable for molecular methods based on polymerase chain reaction (PCR); however, goat sperm DNA extraction is limited. Thus, the aim of this study was to evaluate three methods to extract DNA from goat sperm for use in PCR. Eight goat semen pools were used for DNA extraction by using the DNeasy Blood & Tissue Kit, phenol-chloroform, and Chelex-100 methods. DNA samples were analyzed spectrophotometrically to determine the DNA concentration and purity, visualized on 0.8% agarose gel, and used at different amounts (150, 100, 50, 10, and 1 ng) for PCR with electrophoresis, followed by 1.5% agarose gel electrophoresis. The quantity of DNA extracted with Chelex-100 was higher (P < 0.05) than that obtained with either the DNeasy Blood & Tissue Kit or the phenol-chloroform method, with the phenol-chloroform method yielding a greater quantity (P < 0.05) than the kit. The DNeasy Blood & Tissue Kit produced a higher (P < 0.05) purity product than the Chelex-100 method, and all samples obtained by the three protocols were positive for DNA, as assessed by electrophoresis. All of the different concentrations of DNA produced by these methods were amplified by PCR, although for DNA produced by the phenol-chloroform method, PCR was only possible after complementary purification. In conclusion, the Chelex-100 method is cheap, secure, simple, fast, and effective, and is a potential tool for extracting goat sperm DNA without limitations in PCR.

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The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133 mg follicle-stimulating hormone (FSH) i.m. in six descending doses at 12-h intervals. The goats received 4.8 µg/kg human recombinant leptin s.c. (leptin group, n = 5) or phosphate-buffered saline (PBS) (control group, n = 5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6 days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick-end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E2 ) and progesterone (P4 ) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p < 0.005), fewer embryos classified as degenerated (p < 0.001) and fewer TUNEL-positive cells/blastocyst (p < 0.001). The number of transferable embryos was positively correlated with E2 concentrations on day of oestrus (r = 0.562; p < 0.01) and P4 concentrations on the day of embryo collection (r = 0.912; p < 0.001). We concluded that in vivo leptin administration during FSH treatment improved embryo quality and affected ovarian steroidogenesis in superovulated goats.

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The objective of this work was to study, through ultrasonographic evaluation, changes in testes and epididymides of clinically healthy, peripubertal and pubertal Santa Inês lambs raised in Brazil. Periodic e valuations of weight, biometric characteristics (scrotal circumference, width and length) and ultrasound examinatio ns of the testes and epididymides of 20 lambs were performed between 84 and 280 days old at intervals of 28 days. Scans were performed in the sagittal, transverse, frontal and oblique planes to evaluate the echotexture of the testicular parenchyma and mediastinum and the tail epididymis as well as the thickness and width of the mediastinum testis. The testicular parenchyma demonstrated a homogeneous echogenicity patter n ranging from low to moderate. The echogenicity of testicular parenchyma increased in direct proportion to animal age, being higher in pubertal lambs when compared to prepubertal at the same age. The mediastinum testis was observed in 100% of the evaluated animals, regardless of the scan plane used, and was classified as diffuse or moderately or highly echogenic. Echogenicity and the thickness of the mediastinum testis increased in direct proportion to animal age. The epididymal tail was presented in hypoechoic relation to the testicular parenchyma. Based on these results, it was concluded that ultrasound is useful tool for selection and morphophysiological evaluation of Santa Inês lambs on peripubertal and pubertal phases, when used in combination with other methods such as semen evaluation.

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The objective of this study was to evaluate the influence of testicular disease on sperm morphology. The reproductive tracts of 33 dogs were evaluated clinically and with ultrasound, followed by orchiectomy and harvesting of fluid from the vas deferens to evaluate sperm morphology. A section from each testis was used to conduct histological analyses. Histological changes were noted in 71.2% of testes (47/66). Regardless of dog age, the most frequent pathology was testicular degeneration (80.8%; 38/47), whereas testicular tumors were observed only in adult and old dogs (25.9%; 7/27). Harvesting fluid from the vas deferens for sperm morphology assessment was effective in 87.9% of cases (58/66), and severe testicular degenerative processes induced an increase (P < 0.05) in the percentage of abnormal sperm when compared with normal testes or those with moderate testicular degeneration (special attention given to detached heads). In conclusion, regardless of dog age, breed or origin, a severe testicular degeneration process led to a significant increase in detached heads. Furthermore, the collection of sperm from the vas deferens proved to be an alternative and reliable technique for future research.(AU)

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