Ammonium exposure and pyruvate affect the amino acid metabolism of bovine blastocysts in vitro

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Abstract
The accumulation of ammonium is a major artefact ofin vitroembryo culture. This study has examined ammonium production and potential mechanisms of disposal in preimplantation bovine blastocysts. Embryos were produced byin vitromaturation and fertilisation of oocytes, and cultured in synthetic oviduct fluid containing amino acids and BSA (SOFaaBSA). Ammonium/urea concentrations were determined enzymatically. Amino acid appearance/disappearance ‘profiles’ of single blastocysts were determined at 0, 1.25 and 2.5 mM NH4Cl (with or without 0.33 mM pyruvate), and with or without 10 mM dipicolinic acid (DPCA; a glutamate dehydrogenase (GLDH) inhibitor) or 2 mM amino-oxyacetate (AOA; a transaminase inhibitor). Free ammonium was produced at a rate of 4.281 (±0.362) pmol/embryo/h, while urea production was undetectable. The presence/absence of pyruvate affected amino acid profiles, especially alanine appearance (P< 0.001), glutamate disappearance (P< 0.05) and overall turnover (the sum of appearance and disappearance) (P< 0.001). GLDH inhibition with DPCA had no effect on amino acid overall disappearance, but glutamate disappearance increased, while that of arginine decreased (P< 0.05). The transaminase inhibitor, AOA, depressed turnover (P< 0.05), aspartate and glutamate disappearance, and alanine appearance. Thus, bovine blastocysts release ammonium as free ions or fix them, not as urea, but as alanine, possibly glutamine and, less likely, arginine. An active role for GLDH and transaminases in regulating blastocyst amino acid metabolism was demonstrated.