Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Antioxidantes associados à pressão hidrostática sobre a viabilidade embrionária pós-desvitrificação / Association between antioxidant and hidrostatic pressure on the embryonic viability after

Avaliou-se o efeito de dois antioxidantes no cultivo de embriões pós-desvitrificação, associados ounão à pressão hidrostática (PH) em três experimentos. O primeiro avaliou a interação entre PH e antioxidante(β-mercaptoetanol - BME, cisteamina - CYST e BME + CYST). O segundo, similar ao primeiro, vitrificou osembriões uma hora após passarem ou não pela PH. Os parâmetros foram taxas de eclosão e degeneração com24 e 48 h após passar pela PH (experimento 1) e 12, 24, 48 e 72 h pós-desvitrificação (experimento 2). Oexperimento 3 avaliou a taxa de prenhez de embriões cultivados por 12 h com/sem BME. O primeiroexperimento não demonstrou interação entre os tratamentos. No segundo, os resultados foram similares para BX.O tratamento BME + CYST obteve melhor taxa de eclosão dos BL com 48 e 72 h (76,04%). O mesmo fatoocorreu para a taxa de degeneração às 24 h (BME + CYST = 7,29%; 32,29% controle). Ao transferir os embriões(n = 55), percebeu-se uma similaridade em todos os grupos (38,9% CONT; 16,7% VITRIF e 31,6% BME). Opresente trabalho mostrou que o uso da pressão hidrostática e de BME e CYST não influencia as taxas deeclosão, degeneração e de prenhez de embriões vitrificados.(AU)

This study aimed to evaluate different antioxidants in embryo culture after vitrification, with or withoutthe previous use of hydrostatic pressure (PH). Three experiments were designed to evaluate the interactionbetween PH and antioxidants (β-mercaptoethanol - BME, cysteamine - CYST and BME + CYST) in fresh andvitrified in vitro produced embryos. In experiment 1 hatching and degeneration rates were evaluated at 24 and48 h after passing through the PH and in experiment 2 the same parameters were evaluated at 12, 24, 48 and72 h after heating. The last step of the study evaluated the pregnancy rate of vitrified embryos, cultured for 12 hwith / without BME. The first experiment showed no difference between treatments. The second found similarresults for all parameters evaluated in BX embryos. Note that the BME + CYST treatment obtained a betterhatching rate in BL with 48 and 72 h (76.04%) than the control group (45.83%). The same behavior wasobserved in degeneration 24 h, where the BME + CYST group was 7.29% against 32.29% in the control.However, the pregnancy rates (55 embryo transfers) were not different between control fresh, control vitrifiedand BME groups (38.9%, 16.7% and 31.6%, respectively). This study showed that the use of hydrostaticpressure and antioxidant had no effect on the evaluated parameters.(AU)

Antioxidantes associados à pressão hidrostática sobre a viabilidade embrionária pós-desvitrificação / Association between antioxidant and hidrostatic pressure on the embryonic viability after

Avaliou-se o efeito de dois antioxidantes no cultivo de embriões pós-desvitrificação, associados ounão à pressão hidrostática (PH) em três experimentos. O primeiro avaliou a interação entre PH e antioxidante(β-mercaptoetanol - BME, cisteamina - CYST e BME + CYST). O segundo, similar ao primeiro, vitrificou osembriões uma hora após passarem ou não pela PH. Os parâmetros foram taxas de eclosão e degeneração com24 e 48 h após passar pela PH (experimento 1) e 12, 24, 48 e 72 h pós-desvitrificação (experimento 2). Oexperimento 3 avaliou a taxa de prenhez de embriões cultivados por 12 h com/sem BME. O primeiroexperimento não demonstrou interação entre os tratamentos. No segundo, os resultados foram similares para BX.O tratamento BME + CYST obteve melhor taxa de eclosão dos BL com 48 e 72 h (76,04%). O mesmo fatoocorreu para a taxa de degeneração às 24 h (BME + CYST = 7,29%; 32,29% controle). Ao transferir os embriões(n = 55), percebeu-se uma similaridade em todos os grupos (38,9% CONT; 16,7% VITRIF e 31,6% BME). Opresente trabalho mostrou que o uso da pressão hidrostática e de BME e CYST não influencia as taxas deeclosão, degeneração e de prenhez de embriões vitrificados.

This study aimed to evaluate different antioxidants in embryo culture after vitrification, with or withoutthe previous use of hydrostatic pressure (PH). Three experiments were designed to evaluate the interactionbetween PH and antioxidants (β-mercaptoethanol - BME, cysteamine - CYST and BME + CYST) in fresh andvitrified in vitro produced embryos. In experiment 1 hatching and degeneration rates were evaluated at 24 and48 h after passing through the PH and in experiment 2 the same parameters were evaluated at 12, 24, 48 and72 h after heating. The last step of the study evaluated the pregnancy rate of vitrified embryos, cultured for 12 hwith / without BME. The first experiment showed no difference between treatments. The second found similarresults for all parameters evaluated in BX embryos. Note that the BME + CYST treatment obtained a betterhatching rate in BL with 48 and 72 h (76.04%) than the control group (45.83%). The same behavior wasobserved in degeneration 24 h, where the BME + CYST group was 7.29% against 32.29% in the control.However, the pregnancy rates (55 embryo transfers) were not different between control fresh, control vitrifiedand BME groups (38.9%, 16.7% and 31.6%, respectively). This study showed that the use of hydrostaticpressure and antioxidant had no effect on the evaluated parameters.

In vitro production of Bos taurus indicus embryos with cysteamine

Advanced reproductive techniques use three phases of bovine embryos in in vitro production (IVP), i.e. in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) for a variety of studies. This study assessed the effect of cysteamine, a thiol component, on the embryonic development during IVP. Bos taurus indicus cumulus-oocyte complexes (COC) from ovaries collected at an abattoir were randomly distributed in four groups: control-group (n = 544; without cysteamine), cysteamine in maturation (n = 543), cysteamine in fertilization (n = 540) and cysteamine in the culture medium (n = 557). All COC were matured for 24 h in TCM-199 + 0.01 IU r-hFSH/ml + 0.05 mg LH/ml + 10% fetal calf serum (FCS) at 38.5ºC in 5% CO2 in humidified air. In the cysteamine-maturation group, the TCM-199 medium was supplemented with 150 µm cysteamine (CYS). The IVF (day 0 = fertilization day) was performed for 18-22 h in Fert-Talp medium + heparin + penicillamine, hypotaurine and epinephrine (PHE). The medium of the cysteamine-fertilization group was supplemented with 150 µm CYS. Bos taurus indicus frozen semen was selected by Percoll gradient, and incubated with the oocytes for 18 h. Presumed zygotes were cultured in 400 µl SOFaaci medium + 5% FCS. In the cysteamine-culture group the SOFaaci was supplemented with 150 µm CYS. Embryos were cultured at 5% CO2, 5% O2, 90% N2 and saturated humidity for 8 days. Cleavage rates were 86, 90, 88, and 91%respectively, for control, maturation, fertilization and culture groups. The blastocyst yield at day 7 was 29, 29, 38 and, 36% (P < 0.05) hatched blastocyst yield at day 9 was 21, 25, 27, and 29% (P < 0.05) in the control group and treatments, respectively. Results demonstrated that the addition of cysteamine to the fertilization or culture medium improved blastocyst production.(AU)

In vitro production of Bos taurus indicus embryos with cysteamine

Advanced reproductive techniques use three phases of bovine embryos in in vitro production (IVP), i.e. in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) for a variety of studies. This study assessed the effect of cysteamine, a thiol component, on the embryonic development during IVP. Bos taurus indicus cumulus-oocyte complexes (COC) from ovaries collected at an abattoir were randomly distributed in four groups: control-group (n = 544; without cysteamine), cysteamine in maturation (n = 543), cysteamine in fertilization (n = 540) and cysteamine in the culture medium (n = 557). All COC were matured for 24 h in TCM-199 + 0.01 IU r-hFSH/ml + 0.05 mg LH/ml + 10% fetal calf serum (FCS) at 38.5ºC in 5% CO2 in humidified air. In the cysteamine-maturation group, the TCM-199 medium was supplemented with 150 µm cysteamine (CYS). The IVF (day 0 = fertilization day) was performed for 18-22 h in Fert-Talp medium + heparin + penicillamine, hypotaurine and epinephrine (PHE). The medium of the cysteamine-fertilization group was supplemented with 150 µm CYS. Bos taurus indicus frozen semen was selected by Percoll gradient, and incubated with the oocytes for 18 h. Presumed zygotes were cultured in 400 µl SOFaaci medium + 5% FCS. In the cysteamine-culture group the SOFaaci was supplemented with 150 µm CYS. Embryos were cultured at 5% CO2, 5% O2, 90% N2 and saturated humidity for 8 days. Cleavage rates were 86, 90, 88, and 91%respectively, for control, maturation, fertilization and culture groups. The blastocyst yield at day 7 was 29, 29, 38 and, 36% (P < 0.05) hatched blastocyst yield at day 9 was 21, 25, 27, and 29% (P < 0.05) in the control group and treatments, respectively. Results demonstrated that the addition of cysteamine to the fertilization or culture medium improved blastocyst production.