عامل (هاي ) موثر در كولاپس شدن بلاستوسيست در محيطهاي كشت متوالي

Effective Factors in Blastocyst Collapse in Sequential Culture Media

Resently, sequential culture media (G1.2 and G2.2) have been introduced for blastocyst culture. However,these media in long-term embryo culture, some of hatching (HgB) and hatched (HdB) blastoycsts (the blastocoel gradually becomes smaller and morever, HgB returns to the zona pellucida and some of the blastomers show signs of degeneration). This study was initiated to examine involved factor(s) in this event. So, Rs1 and Rs2 included salts, carbohydrates and glutamine were prepared T6.1 and T62 media as T6 medium. But, their carbohydrates were as Rs1 and Rs2 concentrations, respectively. Nonessential (NEAAs) and essential (EAAs) amino acids, vitamins (vits) and bovine serum albumin (BSA, 4mg/ml, fraction V, free fatty acids) (experiment II, III) or human serum ablumin (Albumin-20) (experiment I, IV) were added to them according to experiment. One-cell mouse embryos (NMRI, out bred) were cultured in the media for 5 days as follow: Experiment I: One-cell embryos were cultured in commercial G12 or Rs1+NEAAs for the first 48h and G22 or Rs2+NEAAs+EAAs+vits for 3 days. Experiment II-1: One-cell embryos were cultured in Rs1 or Rs1+NEAAs for 48h and then cultured in Rs2 and/or Rs2+NEAAs+EAAs for 3 days (group I and 2). Experiment II-2: The medium in the second phase of experiment II (Rs2+NEAAs+EAAs) was renewed after 2 days. Experiment III: One-cell embryos were cultured in T61+NEAAs for 48h and then transferred to T6,2+ NEAAs+ EAAs to culture for 3 days. Experiment IV: One-cell embryos were cultured in R51 +NEAAs and then transferred to Rs2+NEAAs+EAAs. Media in experiments I,III and IV supplemented with BSA and albumin-20 respectively. The results were: experiment I: most embryos (HgB+HdB) were collapsed on the 5th day of culture. Experiment II-1: on the 5th day, there was no collapsed HgB or HdB in group 1 but a few of embryos collapsed in the 2nd group. Experiment II-2: Medium renewalization did not affect blastocyst collapse. Experiment III: The results were the same as experiment 1 (group I) and a few of HgB or HdB were collapsed on the 5th day. Experiment IV: A high percentage of HgB and HdB were collapsed. These results showed that:) human serum albumin (Albumin-20) was a main factor and amino acids were also involved in the blastocyst collapse process,) This process was independent of basic salt solution of sequential culture media, and vitamins and medium renewal did not improve blastocyst collapse.