Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression.
We have previously shown that, while the intrinsic quality of the oocyte is the main factor affecting blastocyst yield during bovine embryo development in vitro, the main factor affecting the quality of the blastocyst is the postfertilization culture conditions. Therefore, any improvement in the quality of blastocysts produced in vitro is likely to derive from the modification of the postfertilization culture conditions. The objective of this study was to examine the effect of the presence or absence of serum and the concentration of BSA during the period of embryo culture in vitro on 1) cleavage rate, 2) the kinetics of embryo development, 3) blastocyst yield, and 4) blastocyst quality, as assessed by cryotolerance and gene expression patterns. The quantification of all gene transcripts was carried out by real-time quantitative reverse transcription-polymerase chain reaction. Bovine blastocysts from four sources were used: 1) in vitro culture in synthetic oviduct fluid (SOF) supplemented with 3 mg/ml BSA and 10% fetal calf serum (FCS), 2) in vitro culture in SOF + 3 mg/ml BSA in the absence of serum, 3) in vitro culture in SOF + 16 mg/ml BSA in the absence of serum, and 4) in vivo blastocysts. There was no difference in overall blastocyst yield at Day 9 between the groups. However, significantly more blastocysts were present by Day 6 in the presence of 10% serum (20.0%) compared with 3 mg/ml BSA (4.6%, P < 0.001) or 16 mg/ml BSA (11.6%, P < 0.01). By Day 7, however, this difference had disappeared. Following vitrification, there was no difference in survival between blastocysts produced in the presence of 16 mg/ml BSA or those produced in the presence of 10% FCS; the survival of both groups was significantly lower than the in vivo controls at all time points and in terms of hatching rate. In contrast, survival of blastocysts produced in SOF + 3 mg/ml BSA in the absence of serum was intermediate, with no difference remaining at 72 h when compared with in vivo embryos. Differences in relative mRNA abundance among the two groups of blastocysts analyzed were found for genes related to apoptosis (Bax), oxidative stress (MnSOD, CuZnSOD, and SOX), communication through gap junctions (Cx31 and Cx43), maternal recognition of pregnancy (IFN-tau), and differentiation and implantation (LIF and LR-beta). The presence of serum during the culture period resulted in a significant increase in the level of expression of MnSOD, SOX, Bax, LIF, and LR-beta. The level of expression of Cx31 and Cu/ZnSOD also tended to be increased, although the difference was not significant. In contrast, the level of expression of Cx43 and IFN-tau was decreased in the presence of serum. In conclusion, using a combination of measures of developmental competence (cleavage and blastocyst rates) and qualitative measures such as cryotolerance and relative mRNA abundance to give a more complete picture of the consequences of modifying medium composition on the embryo, we have shown that conditions of postfertilization culture, in particular, the presence of serum in the medium, can affect the speed of embryo development and the quality of the resulting blastocysts. The reduced cryotolerance of blastocysts generated in the presence of serum is accompanied by deviations in the relative abundance of developmentally important gene transcripts. Omission of serum during the postfertilization culture period can significantly improve the cryotolerance of the blastocysts to a level intermediate between serum-generated blastocysts and those derived in vivo. The challenge now is to try and bridge this gap.
Rizos
D
D
Department of Animal Science and Production, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland.
Gutiérrez-Adán
A
A
Pérez-Garnelo
S
S
De La Fuente
J
J
Boland
M P
MP
Lonergan
P
P
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism*; D002417:Cattle; D002452:Cell Count; D018031:Connexin 43 / Q000235:genetics; D017630:Connexins / Q000235:genetics; D015925:Cryopreservation; D003470:Culture Media; D016895:Culture Media, Serum-Free; D046508:Culture Techniques; D005314:Embryonic and Fetal Development; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007370:Interferon Type I / Q000235:genetics; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D018832:Molecular Chaperones / Q000235:genetics; D018384:Oxidative Stress / Q000235:genetics; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000235:genetics; D011506:Proteins; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000235:genetics; D053606:Receptors, OSM-LIF; D013482:Superoxide Dismutase / Q000235:genetics; D051028:bcl-2-Associated X Protein
D018031:Connexin 43; D017630:Connexins; D003470:Culture Media; D016895:Culture Media, Serum-Free; D007370:Interferon Type I; D015850:Interleukin-6; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D018832:Molecular Chaperones; D011257:Pregnancy Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D051028:bcl-2-Associated X Protein; C020947:interferon tau; C068044:GJB3 protein, human; D013482:Superoxide Dismutase