André Moens

Addition of β-mercaptoethanol or Trolox® at the morula/blastocyst stage improves the quality of bovine blastocysts and prevents induction of apoptosis and degeneration by prooxidant agents

This study was conducted to evaluate the effect of beta-mercaptoethanol (a stimulator of glutathione synthesis) and Trolox (an hydrosoluble analogue of Vitamin E) on bovine embryos cultured from the morula stage (Day 5 post-insemination; pi) under oxidative stress conditions. Culture of embryos with increased doses of Trolox showed a dose-dependent embryotoxicity on Day 8 pi. The use of 400 microM Trolox as well as beta-mercaptoethanol at 100 microM prevented at least partly (P < 0.05) the prooxidant-induced blastocyst degeneration on Day 8. Hatching rates of surviving blastocysts were significantly increased by both antioxidants and beta-mercaptoethanol alone improved their mean cell numbers, which was significant in the ICM (P < 0.05). Analysis of their effect on Day 7 pi showed that both the antioxidants significantly reduced the prooxidant-induced apoptosis and beta-mercaptoethanol diminished the physiological level of apoptosis as well as it stimulated the glutathione synthesis (P < 0.05). In addition, a comparison between in vitro- and in vivo-produced embryos showed that the levels of apoptosis were similar at the same age post-insemination (morulae and blastocysts) but increased steadily with the embryonic age in in vitro ones. In conclusion, beta-mercaptoethanol and Trolox added separately from the morula stage protected embryos against oxidative stress and improved the quality of the resulting blastocysts.

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post coïtum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.

Impact of adding 5.5 mM glucose to SOF medium on the development, metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme‐dispersed male and female transgenic fetal rabbit gonads of 18–22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two‐cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non‐injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (α‐antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co‐transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8 to 16‐cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines.