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Energy substrates and the completion of spontaneous meiotic maturation
- Stephen M. Downs, Elliott D. Hudson
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This study was carried out to examine how different combinations of pyruvate and glucose affect spontaneous meiotic maturation of cumulus-cell-enclosed mouse oocytes (CEO) to metaphase II (MII). Most experiments used an open system in which oocytes were cultured in 1 ml medium in plastic tubes. Initial experiments examined the dose response effects of pyruvate or glucose alone in the presence or absence of 2 mM glutamine. When medium lacked both pyruvate and glucose, more than 91% of the oocytes died in glutamine-free medium during 15 h of culture; viability was restored with the addition of glutamine, but only 11% of the CEO reached MII. In the absence of glutamine, 62–68% of oocytes completed maturation in 0.23–2.3 mM pyruvate, while 44–60% MII was observed in 0.55–27.8 mM glucose. The addition of glutamine to these cultures had a general suppressive effect on the completion of maturation. When glucose was added to pyruvate-containing cultures, the combination of 1 mM pyruvate/5.5 mM glucose was most effective in supporting maturation (about 90% MII), with little effect of glutamine. No further increase in maturation was observed when glucose was increased five-fold (to 27.8 mM). The positive effect of glucose was in part attributed to stimulation of glycolysis and increased production of pyruvate, since a reduced culture volume (8 μl), which allows the accumulation of secreted pyruvate, improved maturation in glucose-containing, but not pyruvate-containing, medium, and FSH, which stimulates glycolysis, increased progression to MII in glucose-containing, but not pyruvate-containing, medium. Yet these results also suggest that glucose has a beneficial effect on maturation apart from simple provision of pyruvate. The pyruvate effect was directly on the oocyte, because denuded oocytes responded more effectively than CEO to this energy substrate. The highest percentage of MII oocytes (96–97%) occurred in microdrop cultures containing glucose but lacking glutamine. These results indicate that glutamine supports oocyte viability but is not an adequate energy source for the completion of spontaneous meiotic maturation and may be detrimental. In addition, while pyruvate and glucose alone can each support meiotic progression of CEO to MII, optimal maturation requires the provision of both substrates to the culture medium when a large volume (1 ml) is used. It is concluded that careful attention to specific energy substrate supplementation and culture volume is important to optimise spontaneous meiotic maturation in vitro.
Lysine requirements and whole-body protein turnover in growing pigs
- D. N. Salter, A. I. Montgomery, Anna Hudson, D. B. Quelch, Rosemary J. Elliott
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- Journal:
- British Journal of Nutrition / Volume 63 / Issue 3 / May 1990
- Published online by Cambridge University Press:
- 09 March 2007, pp. 503-513
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- May 1990
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The influence on protein accretion and whole-body protein turnover of changing dietary protein quality while maintaining constant energy intake was studied by varying the degree of lysine supplementation of a lysine-deficient barley-based diet given to growing pigs. Measurements of nitrogen metabolism and whole-body protein turnover, using both classical and 15N end-product methods following a single dose of lsqb;15N]glycine, were made in 49-kg male pigs given diets containing 109 g lysine-deficient protein/kg supplemented to make them (1) 'deficient', (2) 'adequate' and (3) 'in excess' with respect to lysine. The 15N dose and protein intake values used to calculate amino N flux from the cumulative urinary excretion of 15N in urea and ammonia were corrected respectively for apparent digestibilities of [15N]glycine and total N determined in a separate experiment in pigs fitted with simple ileal cannulas. N retention and biological value were significantly increased by lysine supplementation of the deficient diet to the 'adequate' level, but were not further increased by the higher level of supplementation. Rates of growth paralleled these changes. The poorer biological value of the unsupplemented diet 1 was shown also in a significantly higher excretion of urea N compared with diets 2 and 3. N digestibility was not markedly influenced by the level of lysine supplementation. Both whole-body protein synthesis and degradation increased markedly on 'adequate' supplementation of the diet with lysine, but did not increase further with an excess of lysine. It is concluded that the increase in protein accretion rate observed on supplementation of the diet with lysine was due to a greater increase in the rate of protein synthesis than of degradation, rather than a decrease in degradation rate.