Effect of Modulating Agents on the Induction of Hatching Mechanics in Late Stage Porcine Embryos

Abstract

Hatched blastocyst is crucial for research on the peri-implantation period, and stem cell development, however, hatching mechanobiology still needs to be better understood due to the lack of experimental techniques. Our research aimed to use some modulating agents and timelapse systems to study hatching mechanics in late-stage porcine embryos. Fetal bovine serum (FBS), amino acids (AA), and insulin–transferrin–selenium (ITS) was singly or blended added to in vitro culture to assess their potential on developmental competence of porcine embryos. We found that addition of AA increased expansion and required supply from zygote-stage, ITS impacted detrimentally on porcine embryos. In the presence of FBS, ITS could replace AA in culture medium, and FBS solely was unfulfilled to support growth of late-stage embryos.  Combination of FBS, AA and ITS was sufficient to induce an early hatching, improve quality in terms of Trophectoderm (TE) and Inner cell mass (ICM) cells, and extend culture periods of porcine hatched blastocyst. FBS supportively affected hatching dynamics in porcine blastocyst  by promoting blastocoel expansion, increasing blastomere numbers in same scale of embryo, reducing hatching size and hatching completion time.  Collapse and re-expansion analysis showed that FBS did not modify  autonomous hydraulic control, while Mitomycin treatment in blastocyst suggested FBS induced expansion by other mechanisms rather than increase in cell numbers. Finally, for the establishment of ESC (embryonic stem cells), the result showed ICM isolated from day 7 hatched blastocyst derived in the combination of FBS, AA and ITS yielded higher efficiency in attachment, colony formation, and maintenance than those derived from blastocyst day 8. Therefore, our study demonstrated that early hatched blastocyst at day 7 might be more effective for the establishment of porcine ESCs. In conclusion, combination of FBS, AA and ITS was crucial for late-stage porcine embryo as well as derivation of porcine ESC.

Keywords: Blastocyst hatching, Hatching mechanics, Embryonic stem cells, Fetal bovine serum, Insulin-Transferrin-Selenium.