There is clear clinical need for a novel non-destructive test of oocyte viability. Resazurin enters a cell and is reduced by diaphorases to resorufin, which exits the cell and accumulates in the culture medium, thus providing a measure of viable cell metabolism ¹ .

Follicles (0, 1, 2, 4 or 8 follicles per well) from 6-10 week Swiss mice were added to 50mM resazurin in DMEM+10%FCS in a 96 well plate (n=4). After 12, 14, 16, 18, 20, 22, 24h resorufin was quantified using a spectrophotometer (570nm). Follicles were subsequently stained with CMXRos to identify mitochondria, or α-tubulin-FITC antibody to localise cytoskeletal components, and DAPI to identify the nuclei. Microphotographs of whole follicles were analysed using ImageJ. Alpha-tubulin specific fluorescence per unit area values were subjected to 2-way ANOVA for control (n=9 follicles) v. resazurin exposed (n=6 follicles), and to investigate the effect of follicle size (primary v. secondary) on α-tubulin staining. The limit of detection (LOD) was the OD significantly different from 3x StDev/mean OD of wells containing resazurin alone.

There was a linear relationship between follicle number and resorufin (R²>0.9). The LOD after 14-24 hours was 1 follicle. The α-tubulin and CMXRos staining was located to peripheral cytoplasm of the granulosa cells and oocytes, and DAPI staining was nuclear in all follicles. Preliminary analysis found no significant difference in α-tubulin staining between control and resazurin exposed follicles (P=0.15) or between primary (n=6) and secondary follicles (n=9, P=0.08).

The Resazurin assay was sensitive enough to detect single follicles after 14h, there was a linear increase in resorufin production that reflected cellular metabolism, and preliminary analysis suggests that resazurin did not damage cytoskeletal components. The effect of resazurin on CMXRos staining is being quantified. Future developments will increase sensitivity of the assay and examine application to oocytes and embryos.