Supplementation of culture media with esterfied forms of pyruvate improves mouse embryo development

OBJECTIVE: Examine embryo development following substitution of sodium pyruvate in culture media with ethyl or methyl pyruvate.DESIGN: Prospective study.MATERIALS AND METHODS: Three media were tested to determine effects of pyruvate forms on embryo development. Media 1 contained pyruvate as the sole energy source (lactate-free P1). Media 2 contained pyruvate and lactate (standard P1). Media 3 contained pyruvate, lactate, glucose and amino acids (P1-KSOM +AA). In each media, frozen/thawed 1-cell mouse embryos were randomly distributed between 1 of 4 pyruvate treatment groups: sodium pyruvate, ethyl pyruvate, methyl pyruvate, or no pyruvate. Osmolarity of all media with equalized if necessary by adjusting NaCl levels. Embryos were cultured for 96h in 5% CO2 in air and development graded every 24h. Developmental data were arc sin transformed, analyzed using GLM ANOVA, and statistical differences determined using Bonferroni multiple comparisons test.Table 1Lactate-Free P1 Embryo Development24h Cleavage48h ≥ 8-Cell72h ≥ Morula96h Total Blastocyst96h Hatching BlastocystTotal Blastocyst Cell #Sodium Pyruvate n=49100a22a59a19a0a31.5aEthyl Pyruvate n=49100a50b89b60b7a37.4aMethyl Pyruvate n=49100a49b81b54b8a36.5aNo Pyruvate n=4911b0c0c0a0a–Different superscripts within a column represent statistical differences, P<0.05. Open table in a new tab CONCLUSIONS: These data indicate esterfied forms of pyruvate may benefit mouse embryo development. Current culture media formulations include sodium pyruvate, however, aqueous solutions of pyruvate can be unstable and result in formation of byproducts that interfere with key steps of the TCA cycle, as well as limit ability to scavenge ROS. Ethyl and methyl pyruvate appear to be more stable and have proven superior to sodium pyruvate in protecting somatic cell systems from varying paradigms of redox stress and damage. Furthermore, these esterfied forms of pryuvate are more membrane permeable than sodium pyruvate, and are thus thought to more readily enter the mitochondria and stimulate NADH/NADPH production. Future studies will examine effects of esterfied pyruvate on embryo metabolism. OBJECTIVE: Examine embryo development following substitution of sodium pyruvate in culture media with ethyl or methyl pyruvate. DESIGN: Prospective study. MATERIALS AND METHODS: Three media were tested to determine effects of pyruvate forms on embryo development. Media 1 contained pyruvate as the sole energy source (lactate-free P1). Media 2 contained pyruvate and lactate (standard P1). Media 3 contained pyruvate, lactate, glucose and amino acids (P1-KSOM +AA). In each media, frozen/thawed 1-cell mouse embryos were randomly distributed between 1 of 4 pyruvate treatment groups: sodium pyruvate, ethyl pyruvate, methyl pyruvate, or no pyruvate. Osmolarity of all media with equalized if necessary by adjusting NaCl levels. Embryos were cultured for 96h in 5% CO2 in air and development graded every 24h. Developmental data were arc sin transformed, analyzed using GLM ANOVA, and statistical differences determined using Bonferroni multiple comparisons test. Different superscripts within a column represent statistical differences, P<0.05. CONCLUSIONS: These data indicate esterfied forms of pyruvate may benefit mouse embryo development. Current culture media formulations include sodium pyruvate, however, aqueous solutions of pyruvate can be unstable and result in formation of byproducts that interfere with key steps of the TCA cycle, as well as limit ability to scavenge ROS. Ethyl and methyl pyruvate appear to be more stable and have proven superior to sodium pyruvate in protecting somatic cell systems from varying paradigms of redox stress and damage. Furthermore, these esterfied forms of pryuvate are more membrane permeable than sodium pyruvate, and are thus thought to more readily enter the mitochondria and stimulate NADH/NADPH production. Future studies will examine effects of esterfied pyruvate on embryo metabolism.