176 Oocyte quality assessed by brilliant cresyl blue (BCB) staining recovery by ovum pickup in alpacas (Vicugna pacos)

The quality of oocytes is an important factor for the development of IVF protocols in camelids. Brilliant cresyl blue (BCB) is a colorant that allows the evaluation of the activity of the enzyme glucose-6-phosphate dehydrogenase (G6DPH), which has high levels in growing oocytes and is able to reduce the BCB, making it colorless. The objective of this study was to evaluate the effect of using BCB staining on the selection of competent cumulus–oocytes complexes (COCs), obtained by transvaginal follicular aspiration guided by ultrasonography (ovum pickup, OPU) in alpacas (Vicugna pacos). The hypothesis was that the rate of oocytes selected by BCB would be higher than the traditional selection method. Nonpregnant female alpacas aged 6–8 years with a weight of 60–65 kg were used. The animals were synchronized by follicular ablation of dominant follicles and subjected to an ovarian super-stimulation protocol and subsequent recovery of oocytes by OPU, using the ultrasound unit EXAPAD MINI coupled to a 7.5-MHz micro-convex transvaginal transducer and 19 G needle guide. Follicles were aspired using a vacuum pump with a pressure of 55 mm Hg. A total of 113 COCs were recovered from 253 aspirated follicles (recovery rate, 44.7%). All COCs were evaluated by two different methods: (1) all recovered COCs (113) were evaluated using the traditional method (morphological classification) and (2) the same COCs were stained with BCB. Based on the cytoplasm morphology and layers of cumulus cells, COCs classified as grade 1 or 2 were considered as selected COCs (Control). Next, COCs were stained with 26 μM BCB for 90 min in an incubator at 38.5°C and 100% relative humidity. The COCs were considered BCB+ if the cytoplasm incorporated dyeing and BCB− if the cytoplasm was colorless; only BCB+ were considered selectable (Treatment). The 113 COCs recovered and evaluated according to morphological classification, were classified as G1 (31.0%), G2 (31.0%), G3 (24.8%), and G4 (13.2%), with 62.0% of oocytes being considered selectable (G1 and G2). However, when the same COCs were submitted to BCB staining, 78.8% of oocytes (BCB+) were identified as selectable and 21.2% (BCB−) were non-selectable. It was also shown that from each grade of the morphological classification (G1 to G4), a certain percentage of BCB+ oocytes were obtained: G1 (97.1%), G2 (85.7%), G3 (60.7%), and G4 (53.3%), implying that good-quality oocytes are discarded with the traditional method. In a comparison of the proportion of oocytes selected by the control and treatment method (62.0% and 78.8%, respectively), chi-square testing revealed that the rate of BCB+ oocytes was significantly higher than the number of oocytes selected by the traditional method (P < 0.05). When the change in the proportion of selectable and non-selectable oocytes was shown according to each selection method, the oocytes that changed results were grouped, and the McNemar test showed that this change after BCB use was statistically significant (P < 0.05). The results obtained suggest that BCB staining is a noninvasive and efficient method for selecting good-quality COCs. This method is suitable for use in the development of IVF programs in alpacas because it is a more objective method of selection and, unlike the traditional method, it allows evaluation of the intrinsic quality of oocytes.

This study was supported by Project A21080521 VRIP-UNMSM.