128 CHANGES WITHIN OXYGEN ENVIRONMENT DURING IVF AND IVC IMPROVE BOVINE EMBRYONIC DEVELOPMENT IN VITRO

Abstract

Oxygen concentration during both IVF and IVC affects embryonic development. A low O₂ atmosphere during IVC has been reported to be beneficial for embryos in culture without somatic cells. Similarly, a reduction in spermatozoa (sp) concentration can influence the oxygenation grade of fertilization medium, and O₂ requirements can vary according to the embryonic stage of development. This experiment investigated whether prolonged contact with sperm cells interacts with effects of oxygen tension during the first days of culture. Slaughterhouse bovine COCs matured with Vero-cells in TCM199 with FCS and EGF were co-incubated (CI) with swim-up separated sp. COCs with attached sp were either removed after 2 h and placed in IVF medium without sp up to completion of 18 h (sp-restricted CI), or COCs and sp were left together for 18 h (sp-prolonged CI). Zygotes were cultured in serum-free, B2 medium conditioned with Vero cells (Marquant-Le Guienne et al., 1999 Theriogenology 51, 386), modified as described by Gomez and Diez (2000 Anim. Reprod. Sci. 58, 23–37). According to IVF procedure (sp-restricted or sp-prolonged), embryo culture was made either in 20% O₂ from Day 0 up to Day 3 and in 5% O₂ later on, or in 5% O₂ from Day 0 up to Day 8, in a 2 × 2 factorial design. IVF medium was monitored for Na⁺, K⁺, Ca²⁺, pH, O₂, CO₂ , HCO₃− and lactate (i-STAT analyzer; 5 replicates -R-) at 0 h, 2 h and 18 h without cells (controls), at 2 h in CI and at 18 h both in sp-restricted and sp-prolonged CI. Embryo development was analyzed by CATMOD for effects, and all data were processed by GLM and Duncan test and expressed as LSM ± SE. The presence of cells decreased pO₂ (P < 0.01) in IVF medium at 2 h of CI (154 ± 2.3 without cells v. 144 ± 2.3 with cells). There were differences in pO₂ at 18 h (P < 0.05) between sp-prolonged (140.8 ± 2.3) and sp-restricted CI (148.6 ± 2.3). At 18 h in CI there was K⁺ depletion (P < 0.05), while Na⁺, Ca²⁺, pH and pCO₂ remained invariable throughout. Lactate production increased (P < 0.05) in CI at 2 h and, together with HCO₃₋ (P < 0.01), at 18 h in sp-prolonged CI. Increasing O₂ up to 20% during the first 3 days of culture improved total and medium to late (M + L; early blastocysts excluded) blastocyst rates upon sp-prolonged CI, which is comparable to sp-restricted CI under both % O₂ conditions during embryo culture. Under 5% CO₂ throughout culture, higher expansion rates occurred in sp-restricted CI, suggesting that this system could improve embryonic viability. Metabolic effects can be inferred from differences in pO₂ , lactate and HCO₃₋ , and research will explore the role of oxygen free radicals in these experimental conditions. Grant support: Eureka 2573; CDTI, PROFIT, and FICYT.