Selection of developmentally competent sheep oocytes using the brilliant cresyl blue test and the relationship to follicle size and oocyte diameter

The aim of the present study was to evaluate the relationship between follicle size and oocyte diameter and the use of the brilliant cresyl blue (BCB) test prior to in vitro maturation (IVM) in the selection of competent sheep oocytes for in vitro embryo production (IVEP). Sheep oocytes were exposed to the BCB stain, diluted in Dulbecco s phosphate-buffered saline, modified with 0.4% BSA (mDPBS) for 90 min. Oocytes with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, the percentage of selected oocytes following the BCB test and oocyte diameter were evaluated (13, 26 or 39 M BCB in mDPBS). In Experiment 2, the relationship between follicle size (small: <3 mm, medium: 3–5 mm and large: >5 mm), oocyte diameter and percentage of selected BCB+ oocytes were evaluated. In Experiment 3, the effectiveness of different concentrations BCB to select the oocytes most competent for IVEP, were evaluated. Oocytes were tested in 3 groups: a control (immediately cultured); holding-control (kept in mDPBS for 90 min before being cultured); and a treatment-incubation (incubated with BCB). In Experiment 1, the percentage and diameter of BCB+ oocytes obtained after exposure to the different BCB concentrations were similar. The diameter of the BCB+ oocytes was significantly greater than the diameter of the BCB− oocytes for each concentration (P < 0.01). In Experiment 2, the oocyte diameter and percentage of fully grown (BCB+) oocytes increased with follicular size. The percentage of the BCB+ oocytes from the small (37.9%), medium (53.5%) and large (72.9%) ovarian follicles was respectively lower (P < 0.001), similar (P > 0.05) and higher (P < 0.0001) than those of the BCB− oocytes (62.1, 46.5 and 27.1%, respectively). In addition, the diameter of the BCB+ oocytes recovered from the medium (154.3 ± 5.3 m) and small follicles (150.1 ± 5.3 m) was larger (P < 0.01), than those of the BCB− oocytes (148.1 ± 6.0 and 145.9 ± 5.7 m, respectively). In Experiment 3, BCB+ oocytes yielded more blastocysts than BCB− oocytes, control and holding-control oocytes. However, cleavage and blastocyst (development) rates was not significantly affected by BCB concentration. In conclusion, it can be said that the BCB test may be a useful way of selecting developmentally competent sheep oocytes for increased IVEP success