Effects of retinoids on the in vitro development of Capra hircus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol (RT) and retinoic acid (RA) on the in vitro development of pre-implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus-oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus-oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39 °C in an atmosphere of 5% (v/v) CO(2) in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39 °C in a humidified atmosphere of 5% (v/v) CO(2), 5% (v/v) O(2) and 90% (v/v) N(2). In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO(2) in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 µg/ml RT and 0.5 µm RA to the embryo culture media stimulated (p < 0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre-implantation development of goat embryos and can be used to enhance in vitro embryo production.

Effects of alpha-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured / Efeitos dos antioxidantes alpha-tocoferol e ternatina na morfologia e na ativação de folículos pré-antrais caprinos cultivados in vitro

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2 percent and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15µM, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2 por cento, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α-tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro.

Effect of retinoids and growth factor on in vitro bovine embryos produced under chemically defined conditions.

Experiments were conducted to investigate the beneficial effects of adding retinol (RT) and retinoic acid (RA) to bovine oocyte maturation media and insulin-like growth factor-I (IGF-I) to embryo culture under chemically-defined conditions. In Experiment 1.1, in vitro maturation (IVM) was performed in basic maturation media (bMM) and supplemented with 0.3microM RT or 0.5microM RA. For embryo development presumptive zygotes and embryos were placed in droplets of potassium simplex optimized medium (KSOM). Addition of RT and RA to bMM improved (p<0.05) blastocyst formation as compared with control treatments. In Experiment 1.2, using embryos originating from oocytes previously treated with RT and RA, the presumptive zygotes were placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The number of 2-4-cell stage embryos developing to the blastocyst and expanded blastocyst stages were greater (p<0.05) when embryo culture media was supplemented with IGF-I. In Experiment 2.1, IVM was conducted with bMM+FSH containing 0.3microM RT or 0.5microM RA. For embryo development, presumptive zygotes were placed in droplets of KSOM. Addition of RT or RA to IVM medium also enhanced (p<0.05) blastocyst formation. The supplementation of embryo culture media with IGF-I resulted in a greater number (p<0.05) of 2-4-cell stage embryos developing into blastocysts, expanded blastocysts and hatched blastocysts. In Experiment 2.2, using embryos originating from oocytes previously treated with RT and RA, presumptive zygotes were also placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The supplementation of embryo culture media with IGF-I resulted in a greater (p<0.05) number of 2-4-cell stage embryos developing to the blastocyst, expanded blastocyst and hatched blastocyst stages.

Effects of retinol on the in vitro development of Bos indicus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol on the in vitro development of early embryos of cultured Bos indicus (Expt 1) to the blastocyst stage in medium simplex of optimization (KSOM) or sintetic fluid of oviduct (SOF) or co-cultured (Expt 2) with an oviduct cell monolayer (OCM) in KSOM or SOF. A total of 3149 cumulus-oocyte complexes obtained by aspirating follicles (2-5 mm diameter) from ovaries of slaughtered animals were selected for IVM and incubated in TCM 199 supplemented with 25 mM HEPES at 39 degrees C in air with 5% CO(2) and maximum humidity for 24 h. In vitro fertilization (IVF) was performed in modified defined medium (mDM) medium. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes were randomly allocated to the experimental groups. Zygotes cultured (Expt 1) in KSOM + retinol, KSOM, SOF + retinol and SOF were incubated in maximum humidity at 39 degrees C, 5% CO(2), 5% O(2) and 90% N(2). Zygotes co-cultured (Expt 2) in KSOM + retinol + OCM, KSOM + OCM, SOF + retinol + OCM and SOF + OCM were incubated at 39 degrees C, 5% CO(2). In both experiments media were partially changed 48 h after IVF and unfertilized ova were removed. Afterwards embryos were kept in culture or co-culture for further 9 days. In Expt 1, blastocyst rates (day 7) were 14.6% (KSOM + retinol), 15.8% (KSOM), 16.4% (SOF + retinol) and 15.9% (SOF). In Expt 2, the blastocyst rates (day 7) were 25.4% (KSOM + retinol + OCM) 14.2% (KSOM + OCM), 24.3% (SOF + retinol + OCM) and 15.9% (SOF + OCM). The same influence profile of retinol was observed in the formation of the expanded (day 9) and hatched (day 11) blastocysts. The results obtained in Expt 2 demonstrated that the addition of 0.28 microg/ml retinol to the embryo culture media used in this study had a significant (p < 0.05) positive effect on bovine early embryonic development, under the conditions tested, and can be used to enhance in vitro embryo production.