EXOGENOUS gamma-GLUTAMYL CYCLE COMPOUND SUPPLEMENTATION TO IN VITRO MATURATION MEDIUM AND THE EFFECTS ON SUBSEQUENT IN VITRO FERTILIZATION AND CULTURE PARAMETERS OF PORCINE OOCYTES AND THEIR IMPACT ON EMBRYO VIABILITY
Whitaker, Brian Daniel
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High concentrations of intracellular glutathione enhance the in vitro production of porcine embryos. Six experiments were conducted to study the effects of varying concentrations of different supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and in vitro culture (IVC), and evaluate subsequent embryo viability. In Exp. 1, 2, 3, and 4, porcine oocytes were matured in either 3.3 mM cysteine, 150 Î¼M cysteamine, 3.3 mM cysteine and 150 Î¼M cystemaine; 1.0 mM glycine, 2.5 mM glycine, 5.0 mM glycine; 1.0 mM L-glutamate, 2.5 mM L-glutamate, 5.0 mM L-glutamate; and 3.3 mM L-a-aminobutyrate, 25 Î¼M Î²-mercaptoethanol, 3.3 mM cysteine and 25 Î¼M Î²-mercaptoethanol, or 3.3 mM L-a-aminobutyrate and 25 Î¼M Î²-mercaptoethanol. After IVM (44 h), concentrations of intracellular glutathione (GSH) were determined using a colorimetric assay based on absorbency. The supplements that elicited significantly (P < 0.05) the greatest increase in GSH concentrations were 3.3 mM cysteine, 1.0 mM L-glutamate, 3.3 mM L-a-aminobutyrate, and 3.3 mM L-a-aminobutyrate with 25 25 Î¼M Î²-mercaptoethanol. In Exp. 5, oocytes matured with 3.3 mM L-a-aminobutyrate and 25 Î¼M Î²-mercaptoethanol had a significantly less (P < 0.05) occurrence of polyspermy and greater occurrence of MPN formation during IVF compared to the other treatment groups and a significantly greater percentage (P < 0.05) of embryos reaching the 2 cell developmental stage by 48 h post-IVF and blastocyst stage of development by 144 h post-IVF compared to the other treatment groups. In Exp. 6, treatment had no effect on the time of cell death. The times at which embryo mortality was significantly the greatest (P < 0.05) were located within the middle of IVC. The approximate time of the onset of cell death occurred between 24 to 42 h post-IVF with the greatest occurrence around 36 h. These results suggest that supplementing 3.3 mM L-a-aminobutyrate and 25 Î¼M Î²-mercaptoethanol into the IVM medium 1) increases intracellular GSH concentrations by the end of IVM, 2) decreases the occurrence of polyspermy during IVF, 3) increases the MPN formation during IVF, and 3) increases embryo development parameters during IVC. Supplementation to the maturation media does not have an effect on cell death during embryo development. The onset of cell death appears to occur between 24 to 42 h post-IVF with the greatest occurrence around 36 h post-IVF. In order to increase the success of in vitro derived porcine embryos and offspring, the basic fundamentals of the system need to be fully understood.
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