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

Abstract

The accumulation of ammonium is a major artefact of in vitro embryo culture. This study has examined ammonium production and potential mechanisms of disposal in preimplantation bovine blastocysts. Embryos were produced by in vitro maturation 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 NH 4 Cl (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 (6 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.

Improved felid embryo development by group culture is maintained with heterospecific companions

“…However, the efficacy of growth factors derived from various species that have been used successfully as media supplements indicates that species differences are not significant [77][78][79]. Similarly, ammonium production and susceptibility is likely equivalent among species [76,80]. There may be a difference in the species sensitivity to reactive oxygen, with mouse embryos expressing a wider array of antioxidant genes than cattle embryos [74].…”

Fluctuations in bovine ovarian follicular fluid composition throughout the oestrous cycle

“…The bovine oocyte maturation medium used was based on bicarbonate-buffered TCM-199 supplemented with fetal calf serum (Sigma, Poole, Dorset, UK), as used routinely in our laboratories (original protocol from C Galli & G Lazzari; Laboratorio di Tecnologie della Riproduzione (LTR-CIZ), Via Porcellasco 7/f, 26100 Cremona, Italy) (Gopichandran & Leese 2003, Orsi & Leese 2004a. Details of its supplements are given in Table 1.…”

“…These may be beneficial by regulating metabolism (as shown for early mouse embryos (Lane & Gardner 1998)) and protein synthesis (Kuran et al 2002), and by conferring protection against osmotic shock (Lane 2001) and oxidative stress (Lindenbaum 1973). However, they also result in ammonium production as a result of spontaneous degradation and catabolism (Lane & Gardner 1994, Orsi & Leese 2004a, although this is unlikely to accumulate to toxic levels over the 24-h maturation period, since bovine OCCs are relatively tolerant to ammonium (Hammon et al 2000a,b). The ratio of different amino acids may also be important for the oocyte.…”

Improved blastocyst formation with reduced culture volume: comparison of three different culture conditions on 1128 sibling human zygotes

“…Contrarily, in small volumes, embryos are more likely to be exposed to embryotoxic metabolic products (eg. ammonium) which progressively accumulate during embryo culture and which can cause developmental delay or arrest [15][16][17]. Hence, it is important to find an optimal medium volume to balance the right culture volume balancing the beneficial effect of autocrine factors and the negative effect of embryotoxic substances.…”