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)

Endocrine and metabolic differences between Bos taurus and Bos indicus cows and implications for reproductive management

Based on the considerable differences in ovarian morphology and function, as well as circulating hormones and metabolites between Bos indicus (B. indicus) and Bos taurus (B. taurus), researchers are using this acquired knowledge to optimize protocols for fixed-time artificial insemination (FTAI), and production of in vivo derived embryos by multiple ovulation or by in vitro embryo production (IVP). In B. indicus, at the time of follicle deviation, the dominant follicle is smaller and acquires ovulatory capacity at a smaller diameter than B. taurus. Moreover, despite ovulating smaller follicles and having smaller corpora lutea (CL), circulating concentrations of estradiol (E2) and progesterone (P4) are greater in B. indicus than B. taurus. These physiological differences may be related to greater circulating cholesterol, insulin and IGF1 in B. indicus than in B. taurus. For both genetic groups there is a negative relationship between circulating P4 and ovulatory response to the first GnRH treatment of a fixed-time AI (FTAI) protocol. Moreover, despite lower clearance rates of steroid hormones in B. indicus than B. taurus, the dose of 2 mg estradiol benzoate seems to be the most effective either for Nelore (B. indicus beef), Angus (B. taurus beef), or Holstein (B. taurus dairy) cows at the initiation of an E2/P4-based FTAI protocol to optimize synchronization and pregnancy per AI (P/AI). Several studies have shown that only one recommended dose of PGF2α at a FTAI protocol may be insufficient for adequate luteolysis in B. indicus and B. taurus. When submitted to multiple ovulation and embryo transfer, B. indicus cows and heifers need less FSH than B. taurus to achieve superovulation. Moreover, IVP has been more successful in B. indicus than B. taurus due to greater antral follicle count and anti-mullerian hormone, and better oocyte quality.

Endocrine and metabolic differences between Bos taurus and Bos indicus cows and implications for reproductive management

Based on the considerable differences in ovarian morphology and function, as well as circulating hormones and metabolites between Bos indicus (B. indicus) and Bos taurus (B. taurus), researchers are using this acquired knowledge to optimize protocols for fixed-time artificial insemination (FTAI), and production of in vivo derived embryos by multiple ovulation or by in vitro embryo production (IVP). In B. indicus, at the time of follicle deviation, the dominant follicle is smaller and acquires ovulatory capacity at a smaller diameter than B. taurus. Moreover, despite ovulating smaller follicles and having smaller corpora lutea (CL), circulating concentrations of estradiol (E2) and progesterone (P4) are greater in B. indicus than B. taurus. These physiological differences may be related to greater circulating cholesterol, insulin and IGF1 in B. indicus than in B. taurus. For both genetic groups there is a negative relationship between circulating P4 and ovulatory response to the first GnRH treatment of a fixed-time AI (FTAI) protocol. Moreover, despite lower clearance rates of steroid hormones in B. indicus than B. taurus, the dose of 2 mg estradiol benzoate seems to be the most effective either for Nelore (B. indicus beef), Angus (B. taurus beef), or Holstein (B. taurus dairy) cows at the initiation of an E2/P4-based FTAI protocol to optimize synchronization and pregnancy per AI (P/AI). Several studies have shown that only one recommended dose of PGF2α at a FTAI protocol may be insufficient for adequate luteolysis in B. indicus and B. taurus. When submitted to multiple ovulation and embryo transfer, B. indicus cows and heifers need less FSH than B. taurus to achieve superovulation. Moreover, IVP has been more successful in B. indicus than B. taurus due to greater antral follicle count and anti-mullerian hormone, and better oocyte quality.(AU)

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)

Update and overview on assisted reproductive technologies (ARTs) in Brazil

The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.

Update and overview on assisted reproductive technologies (ARTs) in Brazil

The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.(AU)

Comparison of CapriPure® and Percoll® density gradients for sperm separation of frozen-thawed goat spermatozoa

Frozen - thawed semen from six Boer bucks was pooled and used to compare the recently available commercial product CapriPure ® with Percoll ® for sperm quality . Sperm quality parameters such as sperm motility, concentration, membrane integrity (PI/CFDA), acrosome integrity (FITC - PNA) and mitochondrial activity (JC - 1) were evaluated before and after sperm processing using CapriPure ® and Percoll ® density gradient s . There was a significant r eduction (P < 0.05) in the percentage of spermatozoa with intact acrosomes following Percoll ® gradient centrifugation. However , t here was a significant increase in the percentage of spermatozoa with high mitochondrial membrane potential in both CapriPure ® (P < 0.01) and Percoll ® (P < 0.05) density gradient centrifugation methods . The samples selected using the CapriPure ® gradient achieved higher numerical values of membrane integrity, acrosome integrity as well as high mitochondrial membrane potential and s howed a tendency for improvement of sperm parameters compared with the Percoll ® gradient, suggesting that CapriPure ® is a good alternative to Percoll ® for goat sperm separation . However, further studies are needed to assess the fertilizing capacity of sper matozoa following the use of the CapriPure ® gradient.

Comparison of CapriPure® and Percoll® density gradients for sperm separation of frozen-thawed goat spermatozoa

Frozen - thawed semen from six Boer bucks was pooled and used to compare the recently available commercial product CapriPure ® with Percoll ® for sperm quality . Sperm quality parameters such as sperm motility, concentration, membrane integrity (PI/CFDA), acrosome integrity (FITC - PNA) and mitochondrial activity (JC - 1) were evaluated before and after sperm processing using CapriPure ® and Percoll ® density gradient s . There was a significant r eduction (P < 0.05) in the percentage of spermatozoa with intact acrosomes following Percoll ® gradient centrifugation. However , t here was a significant increase in the percentage of spermatozoa with high mitochondrial membrane potential in both CapriPure ® (P < 0.01) and Percoll ® (P < 0.05) density gradient centrifugation methods . The samples selected using the CapriPure ® gradient achieved higher numerical values of membrane integrity, acrosome integrity as well as high mitochondrial membrane potential and s howed a tendency for improvement of sperm parameters compared with the Percoll ® gradient, suggesting that CapriPure ® is a good alternative to Percoll ® for goat sperm separation . However, further studies are needed to assess the fertilizing capacity of sper matozoa following the use of the CapriPure ® gradient.(AU)