P-309 High-viscosity oil overlay provides less protection against media evaporation in extended dry incubation conditions compared with conventional light oil but a reduced heat loss
Authors: Murray L , Patel N , Ozkavukcu S , Lopes F 
Published: June 2023

Abstract

Study question
Does high-viscosity oil (HVO) improve stability on osmolality, temperature, pH, Na+ concentration, and mouse embryo development compared with conventional light oil (CLO)?

Summary answer
In dry incubation condition, osmolality and Na+ concentration were significantly higher in embryo culture media overlaid with either oils, but HVO was worst than CLO.

What is known already
Suboptimal in-vitro culture conditions affect embryo quality and viability. Overlaying culture media with oil is used to maintain a stable optimal culture environment, including reduction of evaporation. Despite dry incubators are known to increase evaporation, they have become more common, especially with the widespread use of time-lapse imaging. The risk of excessive evaporation with the consequent impact on osmolality is even more significant with the promoted usage of undisturbed extended embryo culturing. Novel products (HVO) therefore been developed and recently commercialised with the aim of reducing such effect. Nonetheless, no independent data are available to determine their superiority over CLO.

Study design, size, duration
Culture media dishes overlaid with CLO or HVO were incubated in dry and humidified conditions while mimicking extended culture conditions in four independent experimental groups. Changes in osmolality, pH and Na+ concentrations over three days were measured. Lipid peroxidation and mouse embryo development were recorded to assess toxicity. Additionally, the ability of different oils to maintain the temperature in the culture drops when culture dishes were removed from the incubator was compared.

Participants/materials, setting, methods
Standard embryo culture media drops overlayered with HVO or CLO were incubated in two equally calibrated, identical incubators providing dry or humid conditions. Osmolality (Day1, D3, D5 measured by freezing-point depression osmometer), temperature drop rate at room temperature (T-type thermocouple sensor probe), pH and [Na+] (hand-held blood gas analyser), mouse embryo assay (MEA, 75 embryos from naturally mated CD1 female mice (N = 5), and lipid peroxidation end-product levels (MDA, TBARS assay) were compared among groups.

Main results and the role of chance
The osmolality of the media incubated in humidified conditions did not show a significant change throughout the 5-day experiment, regardless of the type of oil used. However, in dry conditions, a significant daily increase in osmolality was evident; changes were more prominent under HVO (D1, 278.13 ± 0.82; D3, 286.00 ± 1.51; D5 289.86 ± 1.17 mOsm/kg H2O ± SEM. p ≤ 0.001). The osmolality of the media overlaid by the same type of oil raised between different incubators, and reached a significantly higher value on D5. [Na+] values were consistent with the osmolality changes. The recovery time of the temperature on the heated stage to 37 °C was similar in CLO- and HLO-overlaid media (1:07 ± 1:02 min and 2:00 ± 0.7 min, respectively). The temperature drop rate after media were kept at ambient temperature was similar in the first 4 min. By the end, the temperature difference reached a significance between CLO and HVO overlays (32.4 ± 0.4 °C and 32.7 ± 0.4 °C respectively, p = 0.045). No significant differences were found in pH, lipid peroxidation or mouse embryo development between the study experimental groups.

Limitations, reasons for caution
The embryos for MEA were obtained without hyperstimulation, resulting in a small sample size for the study. The exact timing of fertilisation was impossible to detect after mice mating, which caused inconsistency in embryo grading.

Wider implications of the findings
Dry incubators may determine suboptimal embryo culture conditions. A newly developed heavy oil proved to be inferior to conventional light oil in maintaining osmolality stable. Further research should be carried out in engineering systems that limit culture media evaporation or support undisturbed embryo culture in humidified conditions.

Trial registration number
not applicable