Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183???1192; Takahashi and First 1992 Theriogenology 37, 963???978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 ??g mL???1 FSH, 5 ??g mL???1 LH, 0.8 mM L-glutamine, and 50 mg mL???1 gentamicin. Mature COC were fertilized in Tyrode???s modified medium, with 30 mg mL???1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose???phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P < 0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5 mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5 mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125 mM compared with 0.5 mM (P < 0.05) and 1.5 mM (P < 0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5 mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.

Effect of Glyceraldehyde-3-phosphate during bovine in vitro embryo culture

DI FRANCESCO, SERENA;BOCCIA, LUCIA;LONGOBARDI, VALENTINA;GASPARRINI, BIANCA
2011

Abstract

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183???1192; Takahashi and First 1992 Theriogenology 37, 963???978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 ??g mL???1 FSH, 5 ??g mL???1 LH, 0.8 mM L-glutamine, and 50 mg mL???1 gentamicin. Mature COC were fertilized in Tyrode???s modified medium, with 30 mg mL???1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose???phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P < 0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5 mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5 mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125 mM compared with 0.5 mM (P < 0.05) and 1.5 mM (P < 0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5 mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11588/372532
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