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(133) EXOGENOUS PROTEIN REDUCES AMMONIUM ION FLUX FROM IN VITRO-PRODUCED BOVINE BLASTOCYSTS. Berg, Debra1, Beaumont, Susan1, Thompson, Jeremy2, Phung, H.3, Dunlop, James3, 1 AgResearch, Hamilton, New Zealand2 Reproductive Medicine Unit, Adelaide, Australia3 AgResearch, Palmerston North, New Zealand ABSTRACT- Bovine blastocysts actively take up exogenous protein. Replacing albumin (BSA) with polyvinyl-alcohol (PVA) during in vitro culture results in: decreased developmental efficiency, increased pyruvate uptake, decreased pyruvate oxidation, differential gene expression and reduction in total protein content of embryos. We hypothesize that bovine blastocysts cultured in the absence of exogenous protein may degrade significantly more endogenous protein, leading to increased ammonium production compared to embryos cultured with protein. Ammonium is a known teratogen and implicated as a mediator of fetal abnormalities following in vitro culture. To test this hypothesis, IVP-derived zygotes (18 h p.i.) were randomly allocated and cultured in mSOFaa supplemented with either two sources of BSA (Sigma, fatty-acid free or ICPbio, 8 mg/ml) or PVA (1 mg/ml). Blastocysts and expanded blastocysts (n=37) were selected for ammonium measurement on D7 and 8. Ammonium flux was measured using a non-invasive Automated Scanning Electrode Technique (ASET) with an ammonium selective electrode constructed from a glass microcapillary (1 KEY WORDS: ammonium, embryo culture, ASET, PVA |
m tip), filled with an ammonium ionophore (Fluka 09879). Flux was measured at 3 points on the circumference of the embryo: adjacent to, 90o and 180o from the inner cell mass (ICM). Measurements were made in a mannitol-based medium. Embryo cleavage was similar amongst the 3 treatments but more blastocysts were obtained when embryos were cultured in ICP BSA (64% vs. 22%=PVA & 34%=Sigma, respectively, P<0.005). Ammonium efflux was greater in embryos cultured in PVA (1.41 pmol/cm2/s) compared with either BSA treatment (0.46=Sigma & 0.42=ICP, respectively, P<0.05). Ammonium flux was anisotropic, with a greater efflux adjacent to the ICM (P<0.001). Grade 1 blastocysts exhibited greater ammonium efflux at the ICM than grades 2 and 3, regardless of culture treatment (P<0.05). These results clearly show that ammonium efflux from blastocysts occurs and is quantitatively dependent on the presence of exogenous protein. The greater flux associated with the ICM and grade 1 blastocysts may be due to higher cell numbers within the ICM. The data significantly supports our hypothesis that increased ammonium production is a product of protein-free culture conditions.