Effect of different sequential and two‐step culture systems on the development, quality, and RNA expression profile of bovine blastocysts produced in vitro

Abstract

In the present study, we examined the effect of two-step and sequential culture systems on the development, quality, and gene expression profile of bovine embryos generated by in vitro fertilization. Presumptive zygotes were randomly allocated to four culture treatments: (1) KSOM + 0.4% BSA for 3 days, and then KSOM + 5% FBS to day 7 (K-K/FBS); (2) KSOM + 0.1% BSA for 3 days, and then SOF + 5% FBS to day 7 (K-S/FBS); (3) KSOM + 0.1% BSA for 3 days, and then SOF + 0.8% BSA to day 7 (K-S/BSA); and (4) KSOM + 0.4% BSA for 3 days, and then KSOM + 0.8% BSA to day 7 (K-K/BSA). Culture medium had no effect on cleavage rate. However, a significant difference (P < 0.01) was observed with the two-step culture systems, yielding higher rate of blastocysts (37 and 32% for K-K/FBS and K-K/BSA, respectively) compared to sequential culture systems (26 and 28% for K-S/FBS and K-S/BSA, respectively). Embryos cultured in sequential K-S/FBS developed slowly, had a lower hatching rate, fewer cells, and a higher apoptosis rate compared to other treatments. Gene expression analysis showed alterations of DNMT1, OCT-4, and SOD2 in embryos cultured in sequential K-S/FBS and SOD1 in embryos cultured in two-step K-K/BSA. In conclusion, in vitro culture systems may have an impact not just in the developmental potential and quality of the generated embryos but also in the gene expression profile, which suggests that changes in the culture medium composition can modulate global gene expression.

Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT

“…It is necessary to note from these data that each embryo production method showed a different ranking for these genes, with a slightly higher difference for embryos produced by SCNT (Table 2), which could be explained by inefficiencies in the process of nuclear transfer attributable to incomplete or abnormal epigenetic reprogramming (Somers et al ., 2006; Beyhan et al ., 2007; Suzuki et al ., 2008). These differences in the stability of the genes confirm that not only in vitro culture conditions may affect the gene expression patterns during embryogenesis in mammals (Niemann & Wrenzycki, 2000; Lazzari et al ., 2002; Rizos et al ., 2002; Rinaudo & Schultz, 2004; Felmer et al ., 2011), but also the methodological procedure used to generate these embryos, which agrees with previous studies between IVF and SCNT embryos (Zhou et al ., 2008; Ross et al ., 2010). A large variation in M value range between the three different embryo production groups was also observed; particularly the M values of SCNT embryos are lower (0.01 for the most stable gene to 0.752 for the least stable gene), a finding that suggested that these embryos have little variation in their expression patterns compared to IVF and ICSI embryos, respectively.…”

Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT

“…It is necessary to note from these data that each embryo production method showed a different ranking for these genes, with a slightly higher difference for embryos produced by SCNT (Table 2), which could be explained by inefficiencies in the process of nuclear transfer attributable to incomplete or abnormal epigenetic reprogramming (Somers et al ., 2006; Beyhan et al ., 2007; Suzuki et al ., 2008). These differences in the stability of the genes confirm that not only in vitro culture conditions may affect the gene expression patterns during embryogenesis in mammals (Niemann & Wrenzycki, 2000; Lazzari et al ., 2002; Rizos et al ., 2002; Rinaudo & Schultz, 2004; Felmer et al ., 2011), but also the methodological procedure used to generate these embryos, which agrees with previous studies between IVF and SCNT embryos (Zhou et al ., 2008; Ross et al ., 2010). A large variation in M value range between the three different embryo production groups was also observed; particularly the M values of SCNT embryos are lower (0.01 for the most stable gene to 0.752 for the least stable gene), a finding that suggested that these embryos have little variation in their expression patterns compared to IVF and ICSI embryos, respectively.…”

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence

“…After activation, the oocytes were cultured in 50 μL drops of KSOM medium supplemented with 1% ( v / v ) BME essential amino acids, 1% ( v / v ) MEM non-essential amino acids, and 4 mg/mL of fatty acid-free bovine serum albumin (BSA). The culture was carried out at 38.5 °C in a gas mixture of 5% CO 2 , 5% O 2 , 90% N 2, and saturation humidity [75].…”

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos

“…Morphologically expanded blastocysts (200–250 μm in diameter) were used for quality assessment [ 13 , 30 ]. Briefly, expanded blastocysts were fixed and washed twice in PBS/BSA and incubated for 30 min at RT in 4% paraformaldehyde containing 10 μg/ml of Hoechst.…”

Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT

“…It is necessary to note from these data that each embryo production method showed a different ranking for these genes, with a slightly higher difference for embryos produced by SCNT (Table 2), which could be explained by inefficiencies in the process of nuclear transfer attributable to incomplete or abnormal epigenetic reprogramming (Somers et al ., 2006; Beyhan et al ., 2007; Suzuki et al ., 2008). These differences in the stability of the genes confirm that not only in vitro culture conditions may affect the gene expression patterns during embryogenesis in mammals (Niemann & Wrenzycki, 2000; Lazzari et al ., 2002; Rizos et al ., 2002; Rinaudo & Schultz, 2004; Felmer et al ., 2011), but also the methodological procedure used to generate these embryos, which agrees with previous studies between IVF and SCNT embryos (Zhou et al ., 2008; Ross et al ., 2010). A large variation in M value range between the three different embryo production groups was also observed; particularly the M values of SCNT embryos are lower (0.01 for the most stable gene to 0.752 for the least stable gene), a finding that suggested that these embryos have little variation in their expression patterns compared to IVF and ICSI embryos, respectively.…”

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence

“…After activation, the oocytes were cultured in 50 μL drops of KSOM medium supplemented with 1% ( v / v ) BME essential amino acids, 1% ( v / v ) MEM non-essential amino acids, and 4 mg/mL of fatty acid-free bovine serum albumin (BSA). The culture was carried out at 38.5 °C in a gas mixture of 5% CO 2 , 5% O 2 , 90% N 2, and saturation humidity [75].…”

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos

“…Morphologically expanded blastocysts (200–250 μm in diameter) were used for quality assessment [ 13 , 30 ]. Briefly, expanded blastocysts were fixed and washed twice in PBS/BSA and incubated for 30 min at RT in 4% paraformaldehyde containing 10 μg/ml of Hoechst.…”

Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT

“…It is necessary to note from these data that each embryo production method showed a different ranking for these genes, with a slightly higher difference for embryos produced by SCNT (Table 2), which could be explained by inefficiencies in the process of nuclear transfer attributable to incomplete or abnormal epigenetic reprogramming (Somers et al ., 2006; Beyhan et al ., 2007; Suzuki et al ., 2008). These differences in the stability of the genes confirm that not only in vitro culture conditions may affect the gene expression patterns during embryogenesis in mammals (Niemann & Wrenzycki, 2000; Lazzari et al ., 2002; Rizos et al ., 2002; Rinaudo & Schultz, 2004; Felmer et al ., 2011), but also the methodological procedure used to generate these embryos, which agrees with previous studies between IVF and SCNT embryos (Zhou et al ., 2008; Ross et al ., 2010). A large variation in M value range between the three different embryo production groups was also observed; particularly the M values of SCNT embryos are lower (0.01 for the most stable gene to 0.752 for the least stable gene), a finding that suggested that these embryos have little variation in their expression patterns compared to IVF and ICSI embryos, respectively.…”

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence

“…After activation, the oocytes were cultured in 50 μL drops of KSOM medium supplemented with 1% ( v / v ) BME essential amino acids, 1% ( v / v ) MEM non-essential amino acids, and 4 mg/mL of fatty acid-free bovine serum albumin (BSA). The culture was carried out at 38.5 °C in a gas mixture of 5% CO 2 , 5% O 2 , 90% N 2, and saturation humidity [75].…”

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos

“…Morphologically expanded blastocysts (200–250 μm in diameter) were used for quality assessment [ 13 , 30 ]. Briefly, expanded blastocysts were fixed and washed twice in PBS/BSA and incubated for 30 min at RT in 4% paraformaldehyde containing 10 μg/ml of Hoechst.…”