Jean-Magloire Feugang

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.

EXPRESSION OF CU/ZN AND MN SUPEROXIDE DISMUTASES DURING BOVINE EMBRYO DEVELOPMENT: INFLUENCE OF IN VITRO CULTURE

Temporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase–polymerase chain reaction (RT‐PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5‐ to 8‐cell embryos while no transcript was found at the 9‐ to 16‐cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo–derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro‐derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions. Mol. Reprod. Dev. 58:45–53, 2001.

Effects of ovum pick-up frequency and FSH stimulation: a retrospective study on seven years of beef cattle in vitro embryo production.

The aim of this retrospective study was to compare the number of follicles, cumulus oocyte complexes (COCs) and cultured In Vitro Produced (IVP) embryos obtained from 1396 non-stimulated Ovum Pick-up (OPU) sessions on 81 donor animals in a twice weekly OPU scheme. Results were obtained from 640 sessions following FSH-LH superstimulation, on 112 donors subjected to OPU once every 2 weeks. The stimulation protocol started with the insertion of an ear implant containing 3 mg norgestomet (Crestar, Intervet, Belgium) 8 days before puncture (day -8). The dominant follicle was ablated by ultrasound-guided follicle puncture on day -6. On day -3 and day -2, cows were injected with FSH (Ovagen, ICP) twice daily (8 am to 8 pm), i.e. a total dose of 160 mug FSH and 40 mug LG per donor per stimulation cycle. Animals were punctured 48 h after the last FSH injection (day 0). Progesterone implants were removed the next day. Stimulated donor cows were treated with this protocol at 14-day intervals. Follicles were visualized with a Dynamic Imaging ultrasound scanner, equipped with a 6.5 MHz sectorial probe. Follicles were punctured with 55 cm long, 18 gauge needles at an aspiration pressure corresponding to a flow rate of 15 ml/min. Cumulus oocyte complexes were recovered and processed in a routine IVF set-up. Results demonstrate that, expressed per session, FSH stimulation prior to OPU increases production efficiency with significantly more follicles punctured and oocytes retrieved. However, when overall results during comparable 2-week periods are considered (four non-stimulated sessions vs one stimulated), more follicles are punctured and more oocytes are retrieved using the non-stimulated protocol. No significant differences in the number of cultured embryos could be detected, indicating that FSH/LH stimulation prior to OPU might have a positive effect on in vitro oocyte developmental competence as more embryos are cultured with less, presumably better-quality, oocytes.

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.