Medio alternativo para la producción in vitro de embriones bovinos <span class="so-article-trans-title" dir="auto"> Translated title: Alternative medium for in vitro production of bovine embryos </span>

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Abstract

Se evaluó la tasa de desarrollo de blastocistos bovinos producidos in vitro utilizando medio de cultivo convencional para embriones bovinos y medio de cultivo para producción in vitro (PIV) de embriones humanos. Se obtuvieron ovocitos de ovarios de vacas Cebú sacrificadas en matadero. Los folículos ováricos fueron aspirados y los complejos cumulus-ovocitos (CCO) con al menos dos capas de células del cumulus intactas fueron madurados durante 22 a 24 h. Posteriormente, los CCO fueron fertilizados con semen bovino descongelado y coincubados por 20 a 24 h. Los presuntos cigotos fueron cultivados por 48 h en medio KSOM o en medio HTF modificado. En los días 3 y 5 post-fertilización in vitro (FIV), los embriones fueron cambiados a gotas nuevas de KSOM o de IVC-Two/IVC-Three. Para ambos cultivos, en el día 3 post-FIV se determinó la tasa de división y entre los 7 a 9 días post-FIV, se evaluó el desarrollo embrionario a etapa de blastocisto. La tasa de maduración in vitro fue 89,7%. La tasa de división fue 82,3% para ovocitos cultivados en KSOM y de 80,1% para ovocitos cultivados en HTF (P>0,05). La tasa de desarrollo de blastocistos fue 31,6% para embriones cultivados en KSOM y 29,5% para embriones cultivados en HTF/IVC-Two/IVC-Three (P>0,05). En conclusión, las tasas de desarrollo in vitro de blastocistos bovinos fueron similares para los embriones cultivados en un medio convencional para PIV de embriones bovinos y para los embriones cultivados en medios utilizados para PIV de embriones humanos, por lo que ambos tipos de medios pueden utilizarse para la PIV de embriones bovinos con resultados aceptables.

Translated abstract

The objective of this study was to evaluate the blastocyst development rate for in vitro produced bovine embryos using either the conventional culture medium for bovine embryos or culture media used for the in vitro production (IVP) of human embryos. Oocytes were obtained from ovaries of slaughtered Zebu cows. Ovarian follicles were aspirated and the cumulus-oocytes complexes (COC) with at least two layers of intact cumulus cells were matured for 22 to 24 h. Following that, COC were fertilized with frozen-thawed bovine semen and coincubated for 20 to 24 h. The presumptive zygotes were cultured for 48 h in KSOM medium or modified HTF medium. On days 3 and 5, post-in vitro fertilization (IVF) embryos were placed into new drops of KSOM or IVC-Two/IVC-Three. For both culture types, the cleavage rate was determined on day 3 post-IVF and the embryo development rate to blastocyst stage was evaluated on days 7 to 9 post-IVF. In vitro maturation rate was 89.7%. Cleavage rate was 82.3% for oocytes cultured in KSOM and 80.1% for oocytes cultured in HTF (P>0.05). Blastocyst development rate was 31.6% for embryos cultured in KSOM and 29.5% for embryos cultured in HTF/IVC-Two/IVC-Three (P>0.05). In conclusion, in vitro blastocyst development rates were similar for both the embryos cultured in the conventional medium for IVP of bovine embryos and the embryos cultured in media used for the IVP of human embryos, thus, both types of media can be used for the IVP of bovine blastocysts with acceptable results.

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Most cited references 74

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Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

Dimitrios Rizos, P Boland, Adrian Ward … (2002)

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality. Copyright 2002 Wiley-Liss, Inc.

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Large offspring syndrome in cattle and sheep.

Nancy Young, I Wilmut, K Sinclair (1998)

Bovine and ovine embryos exposed to a variety of unusual environments prior to the blastocyst stage have resulted in the development of unusually large offspring which can also exhibit a number of organ defects. In these animals, the increased incidence of difficult parturition and of fetal and neonatal losses has limited the large-scale use of in vitro embryo production technologies commonly used in humans and other species. Four different situations have been identified that result in the syndrome: in vitro embryo culture, asynchronous embryo transfer into an advanced uterine environment, nuclear transfer and maternal exposure to excessively high urea diets. However, programming of the syndrome by all of these situations is unpredictable and not all of the symptoms described have been observed universally. Neither the environmental factors inducing the large offspring syndrome nor the mechanisms of perturbation occurring in the early embryo and manifesting themselves in the fetus have been identified.