Sorting and cryopreservation of goat sperm with or without phenolic compounds / Seleção e criopreservação de espermatozoides caprinos com ou sem compostos fenólicos

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)

Sorting and cryopreservation of goat sperm with or without phenolic compounds / Seleção e criopreservação de espermatozoides caprinos com ou sem compostos fenólicos

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)

(+)-Catechin and (-)-epigallocatechin gallate: are these promising antioxidant therapies for frozen goat semen? / (+)-Catequina e (-)-epigalocatequina galato: essas são promissoras terapias antioxidantes para a congelação de sêmen caprino?

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)

(+)-Catechin and (-)-epigallocatechin gallate: are these promising antioxidant therapies for frozen goat semen? / (+)-Catequina e (-)-epigalocatequina galato: essas são promissoras terapias antioxidantes para a congelação de sêmen caprino?

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)

Avaliação in vitro do sêmen congelado de carneiros com diluidor suplementado com miricetina / In vitro evaluation of ram sperm frozen with extender supplemented with myricetin

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)

Avaliação in vitro do sêmen congelado de carneiros com diluidor suplementado com miricetina / In vitro evaluation of ram sperm frozen with extender supplemented with myricetin

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)

High resveratrol or quercetin concentrations reduce the oscillation index of frozen goat semen / Altas concentrações de resveratrol ou quercetina reduzem o índice de oscilação do sêmen congelado caprino

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)

High resveratrol or quercetin concentrations reduce the oscillation index of frozen goat semen / Altas concentrações de resveratrol ou quercetina reduzem o índice de oscilação do sêmen congelado caprino

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)

Influence of catalase and pre-freezing equilibration on post-thaw semen quality and conception rate in ewes laparoscopically inseminated

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.

Fertilization rate and embryo production of superovulated dairy cows after insemination with non-sorted and sex-sorted semen

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.

Fertilization rate and embryo production of superovulated dairy cows after insemination with non-sorted and sex-sorted semen

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)

Influence of catalase and pre-freezing equilibration on post-thaw semen quality and conception rate in ewes laparoscopically inseminated

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)

Avaliação da IATF em novilhas Girolando utilizando-se diferentes protocolos com sêmen sexado: comunicação breve / Evaluation of Timed Artificial Insemination (TAI) in Girolando heifers using different protocols with sexed semen: short communication

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)

Avaliação da IATF em novilhas Girolando utilizando-se diferentes protocolos com sêmen sexado: comunicação breve / Evaluation of Timed Artificial Insemination (TAI) in Girolando heifers using different protocols with sexed semen: short communication

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.

Canine testicular disorders and their influence on sperm morphology

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.

Canine testicular disorders and their influence on sperm morphology

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)

Testicular and epididymal ltrasonography in Santa Inês lambs raised in Brazil

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.

Efeito da adição de glutationa peroxidase e cisteína ao diluidor de congelação do sêmen equino / Effect of glutathione peroxidase and cysteine addition in an equine frozen semen medium

Foram utilizados ejaculados (n=25) de garanhões para avaliar o efeito de glutationa peroxidase (GPx) e cisteína na viabilidade de espermatozoides congelados. O sêmen foi diluído em Botu Crio, com antioxidantes, e foram formados os grupos: G1, Controle; G2, 1U GPx ; G3, 5U GPx; G4, 0,5mM cisteína; G5, 1mM cisteína. Depois foi envasado em palhetas (0,5mL) e congelado. Após descongelação, 37°C por 30 segundos, alíquotas foram analisadas quanto à integridade de membrana plasmática (IMP) e acrossoma (IAc), potencial de membrana mitocondrial (PMM) e cinética, nos tempos zero (T0) e 60 minutos (T60). GPx 5U e cisteína 0,5mM determinaram maior (P<0,05) IAc em T0 do que em T60. Cisteína 1mM resultou em maior (P<0,05) IAc em T60 do que GPx 1 e 5U e cisteína 0,5mM. O PMM de um garanhão no T60 foi mais alto (P<0,05) do que o de dois garanhões. VCL e VAP foram maiores (P<0,05) no T0 do que no T60 do grupo controle, e um garanhão apresentou, em geral, valores cinéticos mais altos (P<0,05) do que os demais. Conclui-se que a adição de glutationa peroxidase, nas concentrações de 1U e 5U, e de cisteína, nas concentrações de 0,5mM e 1mM, não interferem na integridade de espermatozoides criopreservados de equinos, mas preservam os parâmetros cinéticos de VCL e VAP após 60 minutos de incubação. Ressalta-se, ainda, que o garanhão tem uma forte influência nas características espermáticas pós-congelação.(AU)

Ejaculates (n=25) of horses were used to assess the effect of glutathione peroxidase (GPx) and cysteine on the viability of frozen sperm cells. Semen was extended at Botu Crio with antioxidants, and divided in groups: G1, control; G2, 1 U GPx; G3, 5U GPx; G4, 0.5mM cysteine and G5, 1mM cysteine, packed in 0.5mL straws, and frozen. After thawing (37° C for 30 seconds) samples were analyzed for plasma membrane (IMP) and acrosome integrity (IAc), mitochondrial membrane potential (MMP) and kinematic, at zero (T0) and 60 minutes after (T60). GPx 5U and cysteine 0.5mM increased (P<0.05) IAc at T0, when compared to T60. Cysteine 1mM resulted in a higher (P<0.05) IAc on T60, than GPx 1 and 5U, and cysteine 0.5mM. The PMM from a stallion on T60 was higher (P<0.05) than those of two stallions. In sperm kinematic, VCL and VAP were higher (P<0.05) at T0 compared to T60 for the control group, and one stallion showed larger (P<0.05) kinematic values than other animals. It is concluded that the addition of glutathione peroxidase at concentrations 1U and 5U, and cysteine, at concentrations of 0.5mM and 1mM, does not interfere with the integrity of cryopreserved equine sperm, but preserves the kinetic parameters VCL and VAP after 60 minutes of incubation. It should be noted also that the stallion has a strong influence on sperm characteristics post-freezing.(AU)

Morphometry and histomorphometry of the testis in crossbred pigs fed diets with different protein levels / Morfometria e histomorfometria do testículo em suínos híbridos recebendo dietas com diferentes níveis de proteína

Aiming to evaluate the effect of the diet protein content on testicular parameters in pigs, 21 non-gelded male Dalland pigs were used and randomly divided into three groups. Males belonging to groups G2 and G3 received a diet with crude protein levels of 15% below and above, respectively, in relation to G1 (control). At 210 days of age, animals were castrated, and testis and epididymis were collected for morphometric and histomorphometry analyses. No difference was observed in relation to the total length of seminiferous tubules (G1=3239.9±333,3m; G2=2989.4±171,7m and G3=3059.5±254.9m), population of Sertoli cell (G1=4.7±0.5x10(9); G2=4.3±0.3x10(9) and G3=4.7±0.5x10(9)), population (G1=31.6±5.58x10(9); G2=27.3±4.0x10(9) and G3=26.4±3.9x10(9)) and volume of Leydig cells (G1=1289.3±182.6µm³; G2=1179.1±85.4µm³ and G3=1133.3±37.8µm³) and sperm production (G1=5.9±0.9x10(9); G2=5.6±0.6x10(9) and G3=5.1±0.3x10(9)). Protein levels were sufficient to maintain spermatogenesis in different experimental groups. It can be concluded that the magnitude of variation in levels of protein used in different stages of development was not sufficient to promote significant changes in testicular development and spermatogenesis process in adult animals.(AU)

Avaliou-se o efeito do teor de proteína da dieta sobre características testiculares em suínos, utilizando-se 21 suínos da raça Dalland, distribuídos aleatoriamente em três grupos. Os animais do G2 e G3 receberam dieta com porcentagens de proteína bruta de 15% para mais e para menos, respectivamente, em relação ao G1 (controle). Aos 210 dias de idade, os animais foram orquiectomizados e os testículos e epidídimos foram coletados para análises morfométricas e histomorfométricas. Observou-se efeito significativo da porcentagem de proteína sobre o comprimento e a largura dos testículos, e nenhuma diferença foi identificada em relação ao comprimento total dos túbulos seminíferos (G1=3239,9±333,3m; G2=2989,4±171,7m e G3=3059,5±254,9m), à população de células de Sertoli (G1=4,7±0,5x10(9); G2=4,3±0,3 x10(9) e G3=4,7±0,5x10(9)), à população (G1=31,6±5,58x10(9); G2=27,3±4,0x10(9) e G3=26,4±3,9x10(9)) e ao volume das células de Leydig (G1=1289,3±182,6µm³; G2=1179,1±85,4µm³ e G3=1133,3±37,8µm³) e à produção espermática (G1=5,9±0,9 x10(9); G2=5,6±0,6x10(9) e G3=5,1±0,3x10(9)). Os percentuais de proteína foram suficientes para a manutenção da espermatogênese nos diferentes grupos. Pode-se concluir que a magnitude da variação dos níveis de proteína usada em diferentes fases do desenvolvimento não foi suficiente para promover alterações significativas no desenvolvimento testicular e no processo espermatogênico em animais adultos.(AU)

Efeito da temperatura de descongelação na integridade de espermatozoides criopreservados de cães / The effect of the thawing temperature on the integrity of canine cryopreserved sperm

Avaliou-se a influência da temperatura de descongelação na integridade de espermatozoides criopreservados de cães. Foram utilizados reprodutores das raças Basset Hound (n=3) e Rottweiler (n=3), submetidos a colheitas de sêmen por manipulação peniana. As amostras de sêmen foram descongeladas a 37ºC/1min (G1) ou 70ºC/6s (G2) e avaliadas quanto à motilidade progressiva, vigor e integridade do acrossoma após 0, 30 e 60 minutos de incubação (37ºC), e ultraestrutura espermática imediatamente após a descongelação. Em todos os tempos de incubação, a motilidade progressiva dos espermatozoides descongelados a 70ºC por 6s (74,6%) foi mais alta (P<0,05) que a dos descongelados a 37ºC por 1min (64,6%). O vigor espermático não diferiu (P>0,05) entre os grupos, e o porcentual de gametas com acrossomas íntegros foi maior (P<0,05) nos espermatozoides do G1 do que no G2. Lesões ultraestruturais foram identificadas nos espermatozoides descongelados de ambos os grupos, em maior quantidade nos gametas do G2. Conclui-se que amostras congeladas de sêmen de cães devam ser descongeladas a 37ºC por 1min.(AU)

Aiming to evaluate the influence of the thawing temperature on the viability of canine cryopreserved sperm, Basset Hound (n=3) and Rottweiler (n=3) dogs were used, submitted to semen collected through manual manipulation. Semen samples were thawed at 37ºC during 1min (G1) or at 70ºC during 6s (G2), and evaluated for progressive motility, vigor and acrosome integrity, after 0, 30 e 60 minutes of incubation (37ºC), and sperm ultrastructure immediately after thawing. In all incubation times, the average of progressive motility was higher (P<0.05) in samples from G2 Group (74.6%) than from G1 (64.6%). Sperm vigor had no difference (P>0.05) between groups, and the percentage of gametes with intact acrosome was higher (P<0.05) on sperm cells from G1 than from G2. Ultrastructural changes were identified on dog sperm from both groups, and were observed in higher quantity in gametes from G2 Group. It can be concluded that samples of frozen dog sperm must be thawed at 37°C for 1min.(AU)