Entration of hydro-gen peroxide (H2O2), 3) glutathione (GSH) content, and 4) blastocyst quality (as defined by the total number of cells and incidence of DNA fragmentation in the blastocyst).Materials and methodsProcedures for in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes The procedures for oocyte collection, IVM, and IVF have been described previously [23,24]. Presumptive zygotes were obtained after 3 h of IVF and cumulus cells were removed by pipetting vigorously. They were subsequently cultured randomly in groups of 50?5 embryos per 500 of the media to which they had been assigned, as described below. Experimental design The basic medium used for embryo culture was glucosefree NCSU-37 [12] containing 4 mg/ml BSA and 50 mercaptoethanol (IVC medium). The basic medium was then modified by supplementation with sodium pyruvate and sodium lactate or various concentrations of glucose. Generally, culture was performed at 38.5 and 5 CO2 under 5 O2 and 90 N2. To test the effects of different atmospheric conditions, embryos were also cultured under 5 CO2 in air (which contains approximately 20 O2). Experiment 1: Effect of glucose concentration on development of IVP porcine embryos To determine the effect of increasing glucose concentration as a sole energy substrate during early embryonic development on the developmental competence of porcine embryos to the blastocyst stage in vitro, presumptive zygotes were cultured from Days 0 to 2 (the day of IVF was defined as Day 0) in IVC medium containing 1.5, 3.5, 5.5, 10, or 20 mM glucose (Gluc-1.5, Gluc-3.5, Gluc-5.5, Gluc10, and Gluc-20 groups, respectively). Zygotes were also cultured in IVC medium containing 0.17 mM pyruvate and 2.73 mM lactate (Pyr-Lac group), the standard medium used in our culture system [23]. Subsequently, embryos in all energy supplement groups were cultured until Day 6 in IVC medium supplemented with 5.5 mM PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25768400 glucose. Experiment 2: Measurement of intracellular H2O2 levels To determine the quantity of H2O2 produced by the embryos that had been cultured in the presence or absence of glucose during the first 2 days of IVC, the relative intensity of H2O2 production in Day 1 (at the one-cell stage; after 20?2 h of IVC) or Day 2 (at the 4-cell stage; after 44?8 h of IVC) embryos in each energy supplement group was measured using 2′,7′ -dichlorodihydrofluorescein diacetate (DCHFDA; Sigma) [25,26]. DCHFDA is membrane permeant and, therefore, is able to diffusePage 2 of(page number not for citation purposes)Reproductive Biology and Endocrinology 2006, 4:http://www.rbej.com/content/4/1/readily into cells. Within the cell, acetate groups are hydrolyzed by intracellular esterase, forming 2′,7′-dichlorodihydrofluorescein (DCHF). DCHF then fluoresces when it is oxidized by H2O2 to 2′,7′-dichlorofluorescein diacetate (DCF). The level of DCF produced within the cells is related to the concentration of peroxide present; thus, its fluorescent emission enables measurement of the cellular peroxide level [25,26]. Embryos in each energy supplement group were incubated for 15 min in IVC medium containing 10 DCHFDA, and then Isoarnebin 4 web washed in fresh IVC medium before being placed on a glass slide and covered with a cover slip. The fluorescence emissions were recorded as JPEG files using a digital camera (DP 12; BX 51; Olympus, Tokyo, Japan) attached to a fluorescent microscope (BX 51; Olympus) after excitation at 480 nm and emission at 510 nm. The recorded fluorescen.
