Abstract

Recently, there has been much debate involving the time necessary to effectively equilibrate an embryo culture system, i.e. dishes, media, and oil. Glutamine present in the culture medium spontaneously deaminates at 37°C to release embryo-toxic ammonium. Additionally, the source of the oil overlay can impact the culture environment. A sub-optimal oil overlay, combined with free glutamine (Gln), could effectively become embryo-toxic over a short time period (<48 h). Therefore, the aim of this study was to determine how times of media equilibration, using various combinations of oil source and type of Gln, affect embryo development. Zygotes were collected from 4-week-old CF1 outbred female mice following superovulation and mating. Embryos were cultured in groups of 10 in 20-µL drops of medium G1.2. Initially, embryos were cultured in one of 4 treatment media. In this 2 × 2 factorial design, the culture medium was pre-equilibrated 18 h prior to embryo retrieval and contained free Gln or the heat-stable dipeptide alanyl-glutamine (AlaGln), combined with an oil source of either Sigma mineral oil (Sigma-Aldrich Corp., St Louis, MO, USA) or Ovoil paraffin oil (Vitrolife, Inc., Englewood, CO, USA). The initial study was then repeated using only the best and worst case groups to determine the effect of incubation time as a variable (either 2 h or 18 h). Blastocyst development and total cell numbers were analyzed after 72 h of culture, and differences between treatments were assessed using Fisher's exact test and Student's t-test. After 18 h of pre-equilibration (n e 300 embryos/treatment), blastocyst development in Ovoil + AlaGln (38.6%) was significantly greater when compared to: Ovoil + Gln: 25.5% (P < 0.01), Sigma + AlaGln: 12.8% (P < 0.01), and Sigma + Gln: 11.9% (P < 0.01). Additionally, the total cell numbers in comparison to Ovoil + AlaGln (44.6 ± 10) were significantly decreased: 35.5 ± 7 (P < 0.001), 34.9 ± 9 (P < 0.001), and 29.9 ± 9 (P < 0.001), respectively. In the second experiment, blastocyst development and total cell number between Ovoil + AlaGln (n = 224) and Sigma + Gln (n = 264) after 18 h of pre-equilibration were: 40.4% vs. 9.9% (P < 0.01) and 46.6 ± 9 vs. 29.4 ± 9 P < 0.001), respectively. However, after 2 h of pre-equilibration, the results between Ovoil + AlaGln (n = 260) and Sigma + Gln (n = 284) were: 42.3% vs. 18.3% (P < 0.01) and 46.9 ± 10 vs. 33.6 ± 6 (P < 0.001), respectively. Therefore, when comparing blastocyst development and total cell number between pre-equilibration times (2 h vs. 18 h), the Ovoil + AlaGln group, 42.3% vs. 40.4% and 46.9 ± 10 vs. 46.6 ± 9, showed no significant differences, respectively. In contrast, the Sigma + Gln group produced significant differences for both blastocyst development, 18.3% vs. 9.9% (P < 0.01), and total cell number, 33.6 ± 6 vs. 29.4 ± 9 (P < 0.05), between pre-equilibration times (2 h vs. 18 h), respectively. Data presented confirm the need for an alternative source of glutamine in embryo culture media. The data also indicate that the source of oil has a profound effect on the experimental outcome. Using the appropriate oil and form of Gln means that media can be safely equilibrated for 18 h.