107 DIFFERENCES IN THE INNER MITOCHONDRIAL MEMBRANE POTENTIAL BETWEEN NON-CULTURED AND CULTURED PORCINE EMBRYOS

Abstract

In comparison to in vivo derived pig embryos, in vitro culture conditions produce embryos with altered metabolic rates of carbohydrates and fatty acids (Romek M et al. 2010 Theriogenology 74, 265–276), which may compromise embryo viability. Because various energy substrates are metabolized via several aerobic pathways leading to generation of the inner mitochondrial membrane potential (ΔΨm), value of ΔΨm is a key indicator of embryo metabolic activity, closely related to oxygen consumption and cellular energy needs. Therefore, the aim of this study was to compare ΔΨm between non-cultured and cultured pig embryos during early development. The non-cultured embryos were obtained from 6-month-old gilts, whereas those derived in vitro were cultured from zygotes to the appropriate stage in North Carolina State University 23 (NCSU-23) medium supplemented with 4 mg mL^–1 of bovine serum albumin. The ΔΨm measurements were carried out on both non-cultured and cultured 4 to 8 cell embryos, morulae, blastocysts and late blastocysts. For this, embryos were labelled with 0.5 μM Mito Tracker Orange CMTMRos (MtOR) for 30 min at 39°C and then with 0.5 μM Mito Tracker Deep Red (MtDR) for 30 min at 10°C. Using a LSM 510 Meta Zeiss confocal microscope, we measured the amounts of fluorescence (I_MtOR and I_MtDR) emitted from embryos and values of ΔΨm were estimated as the I_MtOR/I_MtDR ratios. The results were analysed by ANOVA and Tukey's test. From the zygote to morula stages, ΔΨm remained unchanged and did not differ between developmentally matched non-cultured and cultured embryos (P < 0.001). The value of ΔΨm increased significantly (P < 0.05) from 0.90 ± 0.26 arbitrary units (a.u.) for morulae to 3.92 ± 0.63 and 2.06 ± 0.38 a.u. for non-cultured and cultured early blastocysts, respectively. Whereas the mean value of ΔΨm was almost 2 times higher in non-cultured than in cultured early blastocysts, the mitochondrial membrane potential was statistically similar (P < 0.05) in the in vivo derived (2.10 ± 0.37 a.u.) compared to cultured (1.87 ± 0.30 a.u.) blastocysts. The lower ΔΨm in cultured early blastocysts may be explained by several-fold higher glucose concentration in NCSU-23 medium than in the oviductal fluid. It was reported that high levels of glucose decreases the Krebs cycle metabolism of pyruvate, glutamine, and glucose, and reduces oxidation rates of fatty acids in cultured pig embryos in comparison with in vivo counterparts. Hence, this impaired metabolism reflected by decreased ΔΨm may be responsible for insufficient energy production and reduced developmental competence of cultured early blastocysts. Therefore, because embryo-cavitation is a critical event in pig development, further effort should be focused on proper blastocyst culture.