Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Abstract

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.

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

“…Production of superoxide anion and H 2 O 2 via NADPH oxidase has been described on a rabbit blastocyst surface [ 43 ], and the incubation of mouse embryos with an inhibitor of NADPH oxidase induces a dose-dependent reduction in H 2 O 2 production [ 26 ]. Since activity of PPP was higher at zygotes and embryos at the 2-cell stage compared with later developmental stages [ 9 , 11 ], thus enhanced ROS production in embryos cultured with glucose in this study may be associated with the glucose utilization by the early developmental stage of porcine embryos through the pentose phosphate pathway (PPP). Taken together, results from the present study indicate that at least a part of glucose toxicity may be caused by the formation of ROS in Day 1 embryos via glucose metabolism through PPP, resulting in a reduction of both cleavage (data not shown) and blastocyst formation rates.…”

“…These studies have also shown that glucose-utilizing pathways are active throughout embryonic development, and that glucose does contribute as an energy source [ 7 - 11 ]. Therefore, glucose-containing media are commonly used for producing porcine embryos in vitro [ 9 , 12 , 13 ]. However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ].…”

“…However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ]. Moreover, it has been also postulated that metabolic activity of pig embryos, which is reflected by glucose and pyruvate uptake as well as lactate production, differs depending on the culture medium used [ 9 ].…”

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

“…Production of superoxide anion and H 2 O 2 via NADPH oxidase has been described on a rabbit blastocyst surface [ 43 ], and the incubation of mouse embryos with an inhibitor of NADPH oxidase induces a dose-dependent reduction in H 2 O 2 production [ 26 ]. Since activity of PPP was higher at zygotes and embryos at the 2-cell stage compared with later developmental stages [ 9 , 11 ], thus enhanced ROS production in embryos cultured with glucose in this study may be associated with the glucose utilization by the early developmental stage of porcine embryos through the pentose phosphate pathway (PPP). Taken together, results from the present study indicate that at least a part of glucose toxicity may be caused by the formation of ROS in Day 1 embryos via glucose metabolism through PPP, resulting in a reduction of both cleavage (data not shown) and blastocyst formation rates.…”

“…These studies have also shown that glucose-utilizing pathways are active throughout embryonic development, and that glucose does contribute as an energy source [ 7 - 11 ]. Therefore, glucose-containing media are commonly used for producing porcine embryos in vitro [ 9 , 12 , 13 ]. However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ].…”

“…However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ]. Moreover, it has been also postulated that metabolic activity of pig embryos, which is reflected by glucose and pyruvate uptake as well as lactate production, differs depending on the culture medium used [ 9 ].…”

Advances in Swine In Vitro Embryo Production Technologies

“…2002). It seems that in vitro– derived 2–4 cell embryos utilized less glucose than morulae and blastocysts (Gandhi et al. 2001), where it plays a major role during compaction and blastocyst formation.…”

Glutamine metabolism and interactions with mechanistic target of rapamycin complex 1 in porcine preimplantation embryos

“…In porcine embryos, glucose metabolism minimally increases from the 1-cell to 8-cell stage (0.4 ± 0.1 to 3.1 ± 0.4 pmol/embryo/4h) but notably increases in the compacted morula (49.5 ± 2.7 pmol/embryo/4h) and further by the blastocyst stage (147 ± 12.0 pmol/embryo/4h) (Flood and Weibold, 1988). Subsequent studies have also shown that glucose metabolism increases after compaction with glycolysis becoming a key metabolic pathway, but metabolic activity is significantly decreased in in vitro-produced embryos compared to in vivo counterparts (Gandhi et al, 2001;Swain et al, 2002).…”

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

“…Production of superoxide anion and H 2 O 2 via NADPH oxidase has been described on a rabbit blastocyst surface [ 43 ], and the incubation of mouse embryos with an inhibitor of NADPH oxidase induces a dose-dependent reduction in H 2 O 2 production [ 26 ]. Since activity of PPP was higher at zygotes and embryos at the 2-cell stage compared with later developmental stages [ 9 , 11 ], thus enhanced ROS production in embryos cultured with glucose in this study may be associated with the glucose utilization by the early developmental stage of porcine embryos through the pentose phosphate pathway (PPP). Taken together, results from the present study indicate that at least a part of glucose toxicity may be caused by the formation of ROS in Day 1 embryos via glucose metabolism through PPP, resulting in a reduction of both cleavage (data not shown) and blastocyst formation rates.…”

“…These studies have also shown that glucose-utilizing pathways are active throughout embryonic development, and that glucose does contribute as an energy source [ 7 - 11 ]. Therefore, glucose-containing media are commonly used for producing porcine embryos in vitro [ 9 , 12 , 13 ]. However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ].…”

“…However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ]. Moreover, it has been also postulated that metabolic activity of pig embryos, which is reflected by glucose and pyruvate uptake as well as lactate production, differs depending on the culture medium used [ 9 ].…”

Advances in Swine In Vitro Embryo Production Technologies

“…2002). It seems that in vitro– derived 2–4 cell embryos utilized less glucose than morulae and blastocysts (Gandhi et al. 2001), where it plays a major role during compaction and blastocyst formation.…”

Glutamine metabolism and interactions with mechanistic target of rapamycin complex 1 in porcine preimplantation embryos

“…In porcine embryos, glucose metabolism minimally increases from the 1-cell to 8-cell stage (0.4 ± 0.1 to 3.1 ± 0.4 pmol/embryo/4h) but notably increases in the compacted morula (49.5 ± 2.7 pmol/embryo/4h) and further by the blastocyst stage (147 ± 12.0 pmol/embryo/4h) (Flood and Weibold, 1988). Subsequent studies have also shown that glucose metabolism increases after compaction with glycolysis becoming a key metabolic pathway, but metabolic activity is significantly decreased in in vitro-produced embryos compared to in vivo counterparts (Gandhi et al, 2001;Swain et al, 2002).…”

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

“…Production of superoxide anion and H 2 O 2 via NADPH oxidase has been described on a rabbit blastocyst surface [ 43 ], and the incubation of mouse embryos with an inhibitor of NADPH oxidase induces a dose-dependent reduction in H 2 O 2 production [ 26 ]. Since activity of PPP was higher at zygotes and embryos at the 2-cell stage compared with later developmental stages [ 9 , 11 ], thus enhanced ROS production in embryos cultured with glucose in this study may be associated with the glucose utilization by the early developmental stage of porcine embryos through the pentose phosphate pathway (PPP). Taken together, results from the present study indicate that at least a part of glucose toxicity may be caused by the formation of ROS in Day 1 embryos via glucose metabolism through PPP, resulting in a reduction of both cleavage (data not shown) and blastocyst formation rates.…”

“…These studies have also shown that glucose-utilizing pathways are active throughout embryonic development, and that glucose does contribute as an energy source [ 7 - 11 ]. Therefore, glucose-containing media are commonly used for producing porcine embryos in vitro [ 9 , 12 , 13 ]. However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ].…”

“…However, it also remains unclear whether the metabolic profiles of embryos cultured in glucose-containing medium are more or less reflective of correct in vivo porcine embryo metabolism, since the concentration of glucose commonly used in the culture medium is much higher than that in the oviduct, where in vivo porcine embryos are exposed to 0.17 mM glucose [ 14 ]. Moreover, it has been also postulated that metabolic activity of pig embryos, which is reflected by glucose and pyruvate uptake as well as lactate production, differs depending on the culture medium used [ 9 ].…”

Advances in Swine In Vitro Embryo Production Technologies

“…2002). It seems that in vitro– derived 2–4 cell embryos utilized less glucose than morulae and blastocysts (Gandhi et al. 2001), where it plays a major role during compaction and blastocyst formation.…”

Glutamine metabolism and interactions with mechanistic target of rapamycin complex 1 in porcine preimplantation embryos

“…In porcine embryos, glucose metabolism minimally increases from the 1-cell to 8-cell stage (0.4 ± 0.1 to 3.1 ± 0.4 pmol/embryo/4h) but notably increases in the compacted morula (49.5 ± 2.7 pmol/embryo/4h) and further by the blastocyst stage (147 ± 12.0 pmol/embryo/4h) (Flood and Weibold, 1988). Subsequent studies have also shown that glucose metabolism increases after compaction with glycolysis becoming a key metabolic pathway, but metabolic activity is significantly decreased in in vitro-produced embryos compared to in vivo counterparts (Gandhi et al, 2001;Swain et al, 2002).…”