The effect of different zwitterionic buffers and PBS used for out-of-incubator procedures during standard in vitro embryo production on development, morphology and gene expression of bovine embryos.
The effect of the zwitterionic buffers HEPES, TES and MOPS and of PBS used for out-of-incubator procedures during standard in vitro embryo production on bovine oocytes and embryo development, morphology and on the expression patterns of eight selected genes: Fgf-4, Lama1, Ube2a, Gsta4, Il6, Sod1, Prss11 and Hspb1, was evaluated. All buffers were prepared at a concentration of 10 mM in TALP medium, with the exception of PBS. The total time of oocyte/embryo exposure to each buffer was approximately 41 min. The cleavage rates and number of embryos that developed to > or =8 cells at day 4 were no different among the buffers tested, however, more blastocysts developed at day 7, 8 and 9 in HEPES and MOPS treatments than in PBS and TES (P<0.05). No difference between buffers in total and apoptotic cell number was found. Except for Hspb1 and Ube2a genes, the levels of expression of the six remaining transcripts were higher in in vivo than in in vitro embryos irrespective of buffer used (P<0.05). In addition, higher expression of Hspb1 and lower expression of Ube2a and Lama1 were observed in PBS and TES than in MOPS and HEPES treatments (P<0.05). Expression of Fgf-4 and Gsta4 in the in vitro embryos was lower in PBS than in the remaining three buffers (P<0.05) and the level of expression of the Il6 gene was not affected by any buffer tested but was lower in in vitro than in in vivo derived embryos. Expression of both Sod1 and Prss11 genes in MOPS were at the level of the in vivo embryos. These results showed that the choice of buffer and short exposure time of approximately 41 min, affects mRNA expression of in vitro produced bovine embryos.
Palasz
A T
AT
Ministry of Science and Innovation, Department of Animal Reproduction, INIA, Madrid, Spain. palasz@inia.es
Breña
P Beltrán
PB
De la Fuente
J
J
Gutiérrez-Adán
A
A
D000818:Animals; D017209:Apoptosis; D002021:Buffers; D002417:Cattle / Q000196:embryology*; D004624:Embryo Transfer / Q000662:veterinary; D004622:Embryo, Mammalian / Q000166:cytology; D005307:Fertilization in Vitro / Q000662:veterinary*; D020869:Gene Expression Profiling / Q000662:veterinary; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D006531:HEPES; D009025:Morpholines; D010710:Phosphates; D014325:Tromethamine / Q000031:analogs & derivatives
D002021:Buffers; D009025:Morpholines; D010710:Phosphates; D014325:Tromethamine; C008550:morpholinopropane sulfonic acid; C004551:TES; D006531:HEPES