Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro.
Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.
Yuan
Y Q
YQ
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
Van Soom
A
A
Coopman
F O J
FO
Mintiens
K
K
Boerjan
M L
ML
Van Zeveren
A
A
de Kruif
A
A
Peelman
L J
LJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002417:Cattle / Q000196:embryology / Q000502:physiology*; D002970:Cleavage Stage, Ovum; D003470:Culture Media; D046508:Culture Techniques; D004622:Embryo, Mammalian; D005314:Embryonic and Fetal Development / Q000187:drug effects*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D015870:Gene Expression; D020287:In Situ Nick-End Labeling / Q000662:veterinary; D010100:Oxygen / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D051028:bcl-2-Associated X Protein
D003470:Culture Media; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D010100:Oxygen