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Growing cattle embryos beyond Day 8 - An investigation of media components.

Theriogenology (2020-12-29)
Ekaterina Isaac, Peter L Pfeffer
ABSTRACT

The growth of viable cattle embryos in culture to stages beyond the hatching blastocyst is of interest to developmental biologists wishing to understand developmental events beyond the first lineage decision, as well as for commercial applications, because a lengthening of the culturing time allows more time for diagnostic tests on biopsies, whereas extended survival can be used as a better assay system for monitoring developmental potential. We here report on a novel extended culture medium for embryo growth until embryonic day (Day) 12. We used a non-invasive morphological characterisation system that scored viability, inner cell mass (ICM) grade, hatching and embryo and ICM diameter. The basal medium was based on published uterine fluid concentrations of amino acids, carbohydrates and electrolytes. Addition of fetal bovine serum was necessary and the additive ITSX greatly improved culture success. We tested the inclusion of a seven-growth factor cocktail consisting of Activin A, Artemin, BMP4, EGF, FGF4, GM-CSF/CSF2 and LIF, as well as omission of individual components of the cocktail. In the context of the growth factor cocktail, Artemin and BMP4 provided the greatest benefit, while FGF omission had more positive than negative effects on embryo characteristics. Lastly, replacement of ITSX by B27-additive led to the most successful culture of embryos, in all media permutations.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Heparin sodium salt from porcine intestinal mucosa, Grade I-A, ≥180 USP units/mg, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Kanamycin sulfate from Streptomyces kanamyceticus, powder, BioReagent, suitable for cell culture, suitable for plant cell culture
Sigma-Aldrich
Mineral oil, light oil, suitable for mouse embryo cell culture, BioXtra
Sigma-Aldrich
Cysteamine hydrochloride, ≥98% (titration)
Sigma-Aldrich
HEPES sodium salt, BioPerformance Certified, suitable for cell culture, ≥99.5%
Sigma-Aldrich
Hypotaurine, ≥98% (TLC)
Sigma-Aldrich
Sodium pyruvate, powder, BioXtra, suitable for mouse embryo cell culture
Sigma-Aldrich
D-Penicillamine, 98-101%
Sigma-Aldrich
Medium 199, With Earle′s salts and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
HEPES, BioXtra, suitable for mouse embryo cell culture, ≥99.5% (titration)