Advanced In Vitro Production of Pig Blastocysts Obtained through Determining the Time for Glucose Supplementation

Abstract

Abstract.The objective was to determine the effect of glucose supplementation on development (to the blastocyst stage) of in vitro matured (IVM) porcine oocytes that were either in vitro fertilized (IVF) or electrically activated (EA). Embryos were incubated for 46 or 58 h post insemination (hpi) in an NCSU37-based medium containing 0.17 mM sodium pyruvate and 2.73 mM sodium lactate (IVCPyrLac), and then transferred to an NCSU37-based medium containing 5.55 mM glucose (IVC-Glu) and cultured until Days 6 (Day 0 = day of EA or IVF). The proportions of oocytes that had formed full blastocysts by Day 6 following transfer to IVC-glu at 46 hpi was 23.5 and 41.2% in the IVF and EA groups respectively; these were lower (P<0.001) than the proportions of oocytes that formed full blastocysts after transfer at 58 hpi (60.3 and 78.7%). However, there was no significant difference in total cell number (at Day 6) between embryos transferred at 46 vs 58 hpi. We inferred that in vitroderived pig embryos can efficiently use glucose as an energy source starting at approximately 58 hpi; exposure to glucose at that time enhanced development to the blastocyst stage as well as blastocyst quality. Key words: Pig embryo, Blastocyst, Glucose usage, In vitro fertilization, Activation (J. Reprod. Dev. 50: [71][72][73][74][75][76] 2004) wine are increasingly used in transgenic technologies, including gene manipulation and nuclear transfer; both of these technologies require in vitro production of large quantities of matured oocytes and embryos. Although substantial progress has been ma de in in vitro cult ure, maturation and fertilization (IVC, IVM and IVF, respectively) of porcine oocytes, the overall efficiency of in vitro production of porcine embryos remains relatively low. Several critical aspects should be considered, i.e. variation in oocyte sources and quality of IVM [1][2][3][4][5][6], and increased polyspermy [7,8] and morphological abnormalities [4,9 ] in porcine embryos produced by IVF. Although substantial efforts have been made to optimize culture conditions for preimplantation porcine embryos, the metabolic needs of the embryo are still not well understood. In many mammals, the utilization of glucose increases as pr e im pl an t at io n d ev el op m en t p r og re s s es ; glycolysis becomes the dominant pathway during preimplantation development [10][11][12][13]. In the pig (as in many other mammals), glucose utilization in highest in morulae and blastocysts [13,14], consistent with the energy required for the f o r m a t i o n a n d e x p a n s i o n o f b l a s t o c y s t s . Furthermore, glucose supplementation enhanced in vitro development of pig embryos [4,16,17], with

Effect of Replacement of Pyruvate/Lactate in Culture Medium with Glucose on Preimplantation Development of Porcine Embryos In Vitro

“…Exposure to glucose starting from 48 h to 63 h of culture in the present study (Experiment 2), showed that replacement of pyruvate/lactate with glucose at 58 h of culture, permitted a greater number of embryos to develop to the blastocyst stage, suggesting that porcine IVP embryos can effectively utilize glucose as energy starting at around 58 h of culture. Our results were consistent with those reported by Medvedev et al [14] in which the developmental potency of IVP porcine embryos and the quality of blastocysts obtained depended on when exposure to glucose began. Exposure to glucose starting at 58 h of culture facilitat ed the embry os to devel op to f ull blastocysts, but exposure at an earlier stage resulted in roughly half the rate of full blastocyst formation and a substantial increase in the incidence of malformed and dead embryos [14].…”

“…Our results were consistent with those reported by Medvedev et al [14] in which the developmental potency of IVP porcine embryos and the quality of blastocysts obtained depended on when exposure to glucose began. Exposure to glucose starting at 58 h of culture facilitat ed the embry os to devel op to f ull blastocysts, but exposure at an earlier stage resulted in roughly half the rate of full blastocyst formation and a substantial increase in the incidence of malformed and dead embryos [14].…”

“…With regard to this, it has been shown that the absence of glucose in the later preimplantation stages of bovine embryos caused a decrease in the blastocyst total cell number [15]. Kikuchi et al [10] and Medvedev et al [14] demonstrated that pyruvate and lactate are important energy supplements in porcine, especially for an early embryonic development in vitro, while blastocyst formation requires much energy due to increased protein synthesis levels [17] and to increased activity of the Na + /K + -ATPase to create the blastocoel cavity [18].…”

“…However, it is k n o w n t h a t e x p o s u r e o f e m b r y o s t o h i g h concentrations of glucose during the early e m b r y o n i c s t a g e s c a u s e s d e v e l o p m e n t a l retardation in many species [4][5][6]17]. Kikuchi et al [13] and Medvedev et al [14] suggest that glucose supplementation in early development might cause harmful effects, such as oxidative stress on genome activation or difficulty in overcoming the four-cell block at around Day 2 of IVC in porcine. The toxicity of methylglyoxal, a metabolic by-product o f g l y c o l y s i s h a s b e e n r e p o r t e d t o c a u s e inactivation of the intracellular glutathione peroxidase that is responsible for scavenging free oxygen radicals [21].…”

“…A previous study by us reported that replacement of pyruvate and lactate with glucose, as energy substrate at 48 h of culture enhanced blastocyst formation and the quality of the in vitro p r o d u c e d ( I V P ) p o r c i n e b l a s t o c y s t s [ 1 3 ] . Furthermore our most recent study showed that the replacement at 58 h of culture was better than at 46 h for the development of IVP embryos to the blastocyst stage [14]. However, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the eight-cell stage [10], and although there was a wide variety of developmental embryonic stages at Day 2 (Day 0= day of in vitro fertilization) of culture (two-to eightcell stages with most of them at the late four-cell stage) [13], only replacement at 46, 48 and 58 h after IVC of IVP embryos were compared in those studies.…”