Resumo

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.

Resumo

No primeiro experimento foram utilizados oócitos de ovários de abatedouro distribuídos em seis grupos: 1) Maturação e Cultivo in vitro sem adição de antioxidantes (Ct-Ct); 2) Maturação com 2 M de quercetina (Qc-Ct); 3) Cultivo com 2 M de quercetina (Ct-Qc); 4) Maturação e Cultivo com 2 M de quercetina (Qc-Qc); 5) Maturação com 100 M de cisteamina e Cultivo com de 2 M de quercetina (Cis-Qc) e 6) Maturação com cisteamina (Cis-Ct). Com base no primeiro experimento, os oócitos provenientes de Ovum pick up foram avaliados nos grupos Cis-Qc e Cis-Ct. Os resultados do experimento 1 e 2 para os grupos Cis-Qc e Cis-Ct foram confrontados. A análise estatística considerou significância de 5% nas avaliações. O percentual médio de desenvolvimento dos embriões foi similar entre os grupos Qc-Ct, Ct-Qc, Cis-Qc e Cis-Ct (49,55±5.1), porém superiores aos grupos Ct-Ct e Qc-Qc (42,4±6.1). A taxa média de blastocistos eclodidos foi similar entre os grupos Ct-Ct, Qc-Ct, CisQc e Cis-Ct (60,9±6.3), e estes superior grupo Qc-Qc (54,7±9.7). Para o número médio de células dos blastocistos o grupo Cis-Ct (145±25) foi superior os grupos Cis-Qc, Ct-Ct e Qc-Ct (128±25) e estes superiores os grupos Ct-Qc e Qc-Qc (109±32). No experimento 2 a taxa de clivagem não diferiu entre Cis-Ct (88,8±3.9) e Cis-Qc (87,7±4.5). Mas o percentual de blastocistos foi superior para Cis-Qc (64,6±9.8) em relação a Cis-Ct (56,4±3.6). Enquanto a taxa de prenhez do grupo 2 Cis-Ct (45,1±2.5) foi superior ao Cis-Qc (38,5±3.5). O percentual de blastocisto do experimento 2 (56,4±3.6 e 64,6±9.8%) foram superiores aos do experimento 1 (48,9±3.6 e 52,2±5.1%) para os grupos Cis-Ct e Cis-Qc, respectivamente. Em conclusão, a produção in vitro de embriões suplementada com antioxidante dependendo do grupo foi positiva, mas este benefício não foi confirmado na taxa de prenhez com o uso da quercetina no cultivo in vitro

In the first experiment we used oocytes from ovaries of slaughterhouse distributed in six groups: 1) Maturation and cultivation without the addition of antioxidants (Ct-Ct); 2) Maturation with 2 M of quercetin (Qc-Ct); 3) Cultivation with 2 M of quercetin (Ct-Qc); 4) Maturation and cultivation with 2 M of quercetin (Qc-Qc); 5) Maturation with 100 M of cysteamine and cultivation with of 2 M of quercetin (CisQc) and 6) Maturation with cysteamine (Cis-Ct). Based in experiment 1, the oocytes of Ovum pick up (OPU) were evaluated in Cis-and Cis-Qc CT. for experiment 3 were assessed the results of experiment 1 and 2 for groups Cis-and Cis-Ct Qc. Statistical analysis significance of 5% was considered in reviews. The percentage of embryos was superior to Qc-Ct, Ct-Qc, Cis-and Cis-Ct Qc (49,55±5.1), about Ct-Ct and QcQc (42.4±6.1). The hatching rate was similar to Ct-Ct, Qc-Ct, Cis-and Cis-Ct Qc (60,9±6.3), however the Group Qc-Qc (54,7±9.7). Had the lowest rate of hatching, followed by the Ct-Qc (57.3 ± 11.0). The number of cells in the embryos of the Group Cis-Ct (145±25) was superior to the Cis-Qc groups, Ct-Ct and Qc-Ct (media 128 ± 25). The embryos of Ct-Qc groups and Qc-Qc (media 109 ± 32) had the fewest cell. The rate of cleavage in the oocytes OPU did not differ between Cis-Ct (88.8±3.9) and Cis-Qc (87.7 ± 4.5). But the percentae of blastocyst was superior to Cis-Qc (64.6 ± 9.8) for the Cis-Ct (56.4 ± 3.6). However, the rate of pregnancy blastocyst Cis group- 4 Ct (45.1±2.5) were higher than those of Cis-Qc (38.5±3.5). The percentage of blastocyst OPU oocytes (56.4 ± 3.6 and 64.6 ± 9.8%) were higher than those of slaughterhouse (48.9 ± 3.6 and 52.2 ± 5.1%) independent of the groups evaluated (Cis-and Cis-Qc Ct), respectively. In conclusion, the in vitro production of embryos supplemented with antioxidant depending on the Group was positive, but this benefit has not been confirmed pregnant rate with the use of quercetin on in vitro cultivation.

Resumo

Complexos cumulus-oócito (COC), oócitos desnudos (DO) e DO cocultivados com células do cumulus em suspensão (DO+CC) foram maturados in vitro (MIV) na presença ou ausência de cisteamina (50mM). Observou-se efeito benéfico da cisteamina durante o cultivo de MIV, pois a maturação nuclear no grupo COC cisteamina foi maior do que a do COC controle (P<0,05). No grupo sem a adição de cisteamina, foi observado que a ausência de CC durante o cultivo de MIV prejudicou a maturação nuclear em DO, em relação ao COC (P<0,05), todavia a cisteamina restaurou a capacidade de progressão da meiose em DO, tornando-os semelhantes aos COC (P>0,05). O acoplamento entre oócitos e CC durante MIV demonstrou ser essencial para aquisição da competência do oócito para suportar o desenvolvimento embrionário inicial, pois COC apresentaram maior porcentagem de blastocistos e eclosão quando comparados a DO e DO+CC (P<0,05). A inclusão de cisteamina no cultivo de MIV não restaurou a aquisição da competência em DO e DO+CC, que permaneceram semelhantes aos do grupo-controle (P>0,05). Conclui-se que a cisteamina no meio de MIV melhora as taxas de maturação nuclear em COC e restaura a capacidade de progressão da meiose em DO. Todavia, na concentração utilizada neste estudo, não promove efeito benéfico no desenvolvimento embrionário.

Cumulus-oocyte complexes (COC), denuded oocytes (DO) and DO co-cultured with cumulus cells in suspension (DO+CC) were in vitro matured (IVM) in the presence or absence of cysteamine (50mM). A beneficial effect of cysteamine was observed during IVM, because the nuclear maturation in the COC cysteamine group was higher than in COC control (P<0.05). In the control group, the absence of CC during IVM impaired nuclear maturation in DO when compared to COC (P<0.05), but cysteamine restored the ability of meiosis progression in DO, making them similar to COC (P>0.05). The coupling between oocytes and CC during IVM proved to be essential for the acquisition of oocyte competence to support early embryonic development, as COC had higher percentages of blastocyst and hatching when compared to DO and DO+DC (P<0.05). However, the inclusion of cysteamine in the IVM culture did not restore the acquisition of competence in DO and DO+DC, which remained similar to the control group (P>0.05). It is concluded that cysteamine in the IVM culture improves the nuclear maturation in COC and restores the progression ability of meiosis in DO. However, in the concentration used in this study, cysteamine does not promote a beneficial effect on embryo development.

Resumo

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.