147 ION COMPOSITION OF CULTURE MEDIUM INFLUENCES MITOCHONDRIAL DISTRIBUTION AND BLASTOCYST DEVELOPMENT OF PREIMPLANTATION PORCINE EMBRYOS

Abstract

Elevated intracellular calcium (Ca²⁺) concentrations impair hamster embryo metabolism and viability (Lane M and Bavister B 1998 Biol. Reprod. 59, 1000–1007). Extracellular magnesium (Mg²⁺) regulates intracellular Ca²⁺ by controlling its uptake and release. In the present study, we examined the effects of altering Ca²⁺ and Mg²⁺ ion concentrations in Purdue Porcine Medium (PPM1) on porcine embryo mitochondrial distribution, metabolic (glycolytic and Krebs cycle) activity, and in vitro developmental potential. Cumulus-oocyte complexes collected from abattoir ovaries were matured for 40–42 h, inseminated with 5 × 10⁵ sperm mL⁻¹ for 5 h, and initially cultured in 1 : 0.4 or 2 : 1 ratio of Ca²⁺ to Mg²⁺ (concentrations in mM) at 38.7°C, in 6% CO₂, 10% O₂, balance N₂. At 22–26, 46–50, and 70–74 h post-insemination, 2-, 4-, and 8-cell embryos, respectively, were removed from culture to evaluate mitochondrial distribution (confocal microscopy after tetramethylrhodamine methyl ester staining) and glycolytic and Krebs cycle activity (5-[³H]-glucose and 2-[C¹⁴]-pyruvate, respectively). Remaining embryos were further cultured to determine developmental competence (2 : 1, n = 548; 1 : 0.4, n = 560). Cleavage was assessed on Day 3 (2 : 1, n = 552; 1 : 0.4, n = 560) of culture. All data were analyzed using GLM ANOVA, except mitochondrial distribution data which were analyzed using GLIMMIX. A majority (P < 0.05) of 2-cell (65%, 13/20) and 4-cell (67%, 22/33) embryos cultured in 2 : 1 displayed a homogeneous mitochondrial distribution. More (70%, 21/30; P < 0.05) 8-cell embryos cultured in 2 : 1 had a perinuclear mitochondrial distribution. When cultured in 1 : 0.4, a majority (61%, 14/23; P < 0.05) of 2-cell embryos displayed a cortical mitochondrial distribution, whereas most (P < 0.05) 4-cell (66%, 19/29) and 8-cell embryos (69%, 18/26) displayed a homogeneous distribution. Glycolytic and Krebs cycle activities were similar (P > 0.05) between treatments and across all cell stages examined. Treatment had no effect (P > 0.05) on cleavage or blastocyst total cell number. Unlike hamster embryos, culturing pig embryos in a higher Ca²⁺ concentration resulted in more embryos developing to the blastocyst stage. Culture medium containing 2 mM Ca²⁺ and 1 mM Mg²⁺ best supports in vitro blastocyst development, possibly by supporting a more correct mitochondrial distribution. These results are not mediated via changes in glycolytic or Krebs cycle activity, thus suggesting that another cellular mechanism plays a key role in developmental competence in early pig embryos.

Table 1 
Effects of Ca²⁺:Mg²⁺ on porcine embryonic development and metabolic activity (mean ± SEM).