Bovine Embryo Culture in the Presence or Absence of Serum: Implications for Blastocyst Development, Cryotolerance, and Messenger RNA Expression1

Abstract

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 o...

Differences in developmental competence and gene expression profiles between buffalo (Bubalus bubalis) preimplantation embryos cultured in three different embryo culture media

“…Although BSA has beneficial effect on embryo development, it is difficult to elucidate specific functions of growth factors or other stimulants as BSA is an undefined mixture of compounds. The presence of serum in a standard culture medium alters the pattern of mRNA expression (Wrenzycki et al 1999;Rizos et al 2003), affects embryo development (Gutierrez-Adan et al 2001) and cryotolerance (Rizos et al 2003), is associated with a reduction in pregnancy rate after transfer to recipients (Lazzari et al 2002), and may produce longterm effects on post-natal development and behaviour (Fernández-Gonzalez et al 2007). Chemically defined conditions without serum or serum derived proteins allow more precise observation of effects of growth or other embryo growth factors in a given medium (Giles and Foote 1997;Sirisathien et al 2003;Oyamada et al 2004).…”

“…Studies on the embryonic genome indicate that currently used in vitro embryo development conditions cannot fully mimic in vivo conditions with regard to mRNA expression. During the culture period, the presence of serum (10 %) can induce a number of subtle modifications in blastocyst development, including increased expression of stress responsive genes and decreased expression of embryo quality genes in bovine (Rizos et al 2003;Tavares et al 2008). Similarly, no report is available regarding the efficacy of different embryo culture media to support effective expression of developmentally important genes to increase the efficiency of healthy embryo production in buffalo under in vitro culture.…”

Does serum cause lipid-droplet accumulation in bovine embryos produced in vitro, during developmental days 1 to 4?

“…In previous reports, the addition of serum on days 1 or 2 doesn't seem to produce improvements in the percentage of blastocysts compared to cultures without-serum [10,20,50]. Also, addition of serum to the culture before completing day 1 of development, significantly reduced the proportion of embryos reaching the morula and blastocyst stages compared to the ones cultured with serum since day 3 [45].…”

“…During cryopreservation, larger droplets lead to a reduction of the organization of the cytoplasm and produce irreversible damage to the embryo [24,26]. Thus we do not expect that 2-16 cells embryos tolerance to cryopreservation will be different between embryos cultured with or without serum, as occurs in blastocysts [50]. In contrast, because the average sizes of lipid droplets increase with time we consider that the tolerance to this procedure may be lower on day 4 embryos.…”

“…In embryos cultured in medium supplemented with serum, excess lipids in the cytoplasm would be responsible for the high sensitivity to cooling and low tolerance to cryopreservation [32]. Omission of the serum during the culture [50] and removal of cytoplasmic lipid droplets [17,59] improve cryopreservation tolerance increasing survival rates of in vitro produced bovine blastocysts.…”