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Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.

Karja NW, Kikuchi K, Fahrudin M, Ozawa M, Somfai T, Ohnuma K, Noguchi J, Kaneko H, Nagai T - Reprod. Biol. Endocrinol. (2006)

Bottom Line: Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups.These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos.Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Support Center, Swine and Poultry Feeding Management Laboratory, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan. karja_nwk@yahoo.com

ABSTRACT

Background: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined.

Methods: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6.

Results: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts.

Conclusion: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

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Intracellular GSH contents of Day 1 (A; five replicate trials were carried out) and Day 2 embryos (B; four replicate trials were carried out) cultured in either IVC medium containing 1.5 to 20 mM glucose or pyruvate-lactate. Within each end point, bars with different letters (a, b) are significantly different for 20% oxygen tension treatment (P < 0.05). No significant differences were found in GSH level within each energy substrate supplement group across oxygen tension treatments. The numbers on the bars represent the number of embryos used for the assay.
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Figure 4: Intracellular GSH contents of Day 1 (A; five replicate trials were carried out) and Day 2 embryos (B; four replicate trials were carried out) cultured in either IVC medium containing 1.5 to 20 mM glucose or pyruvate-lactate. Within each end point, bars with different letters (a, b) are significantly different for 20% oxygen tension treatment (P < 0.05). No significant differences were found in GSH level within each energy substrate supplement group across oxygen tension treatments. The numbers on the bars represent the number of embryos used for the assay.

Mentions: Among the energy substrate supplement groups, no significant differences were found in the GSH contents of Day 1 embryos under either oxygen tension (see figure 4A). Also the GSH content in Day 2 embryos did not differ among energy substrate supplement groups under 5% oxygen tension. However, under 20% oxygen tension, only the Day 2 embryos in the 1.5 mM group had a significantly lower GSH content (P < 0.05) than the embryos in the Pyr-Lac group (see figure 4B). Within each energy substrate supplement group, no significant differences were found across oxygen tension treatments in terms of the GSH content of either Day 1 or Day 2 embryos. No interaction was found by ANOVA between the oxygen concentration and the energy supplement groups in intracellular H2O2 production and GSH levels.


Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.

Karja NW, Kikuchi K, Fahrudin M, Ozawa M, Somfai T, Ohnuma K, Noguchi J, Kaneko H, Nagai T - Reprod. Biol. Endocrinol. (2006)

Intracellular GSH contents of Day 1 (A; five replicate trials were carried out) and Day 2 embryos (B; four replicate trials were carried out) cultured in either IVC medium containing 1.5 to 20 mM glucose or pyruvate-lactate. Within each end point, bars with different letters (a, b) are significantly different for 20% oxygen tension treatment (P < 0.05). No significant differences were found in GSH level within each energy substrate supplement group across oxygen tension treatments. The numbers on the bars represent the number of embryos used for the assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1636643&req=5

Figure 4: Intracellular GSH contents of Day 1 (A; five replicate trials were carried out) and Day 2 embryos (B; four replicate trials were carried out) cultured in either IVC medium containing 1.5 to 20 mM glucose or pyruvate-lactate. Within each end point, bars with different letters (a, b) are significantly different for 20% oxygen tension treatment (P < 0.05). No significant differences were found in GSH level within each energy substrate supplement group across oxygen tension treatments. The numbers on the bars represent the number of embryos used for the assay.
Mentions: Among the energy substrate supplement groups, no significant differences were found in the GSH contents of Day 1 embryos under either oxygen tension (see figure 4A). Also the GSH content in Day 2 embryos did not differ among energy substrate supplement groups under 5% oxygen tension. However, under 20% oxygen tension, only the Day 2 embryos in the 1.5 mM group had a significantly lower GSH content (P < 0.05) than the embryos in the Pyr-Lac group (see figure 4B). Within each energy substrate supplement group, no significant differences were found across oxygen tension treatments in terms of the GSH content of either Day 1 or Day 2 embryos. No interaction was found by ANOVA between the oxygen concentration and the energy supplement groups in intracellular H2O2 production and GSH levels.

Bottom Line: Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups.These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos.Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Support Center, Swine and Poultry Feeding Management Laboratory, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan. karja_nwk@yahoo.com

ABSTRACT

Background: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined.

Methods: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6.

Results: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts.

Conclusion: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

Show MeSH
Related in: MedlinePlus