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Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation

Published online by Cambridge University Press:  11 December 2014

Alejandra E. Velasquez ,
Fidel O. Castro ,
Daniel Veraguas ,
Jose F. Cox ,
Evelyn Lara ,
Mario Briones  and
Lleretny Rodriguez-Alvarez
Alejandra E. Velasquez
Affiliation:
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillan, Chile.
Fidel O. Castro
Affiliation:
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillan, Chile.
Daniel Veraguas
Affiliation:
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillan, Chile.
Jose F. Cox
Affiliation:
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillan, Chile.
Evelyn Lara
Affiliation:
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillan, Chile.
Mario Briones
Affiliation:
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillan, Chile.
Lleretny Rodriguez-Alvarez*
Affiliation:
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion. Avenida Vicente Mendez 595, Chillan, Chile.
*
All correspondence to: Lleretny Rodriguez-Alvarez. Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion. Avenida Vicente Mendez 595, Chillan, Chile. Tel: +56 042 2208835. e-mail address: llrodriguez@udec.cl
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Summary

Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7–9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.

Keywords

Bovine embryosDemi-embryosElongationGene expressionSplitting
Type
Research Article
Information
Zygote , Volume 24 , Issue 1 , February 2016 , pp. 18 - 30
Copyright
Copyright © Cambridge University Press 2014 

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