Effect of Brilliant cresyl blue (BCB) staining in oocyte’s maturation and parthenogenetic activation in alpaca (Vicugna pacos) oocytes

Abstract

Oocyte quality is a limiting factor for in vitro embryo production (IVEP). This study aimed to evaluate the brilliant cresyl blue (BCB) staining as a marker for selecting competent oocytes in alpacas, in vitro maturation (IVM), and get blastocyst embryos after parthenogenetic activation (PA). Ovaries from a slaughterhouse were collected and transferred to the laboratory at either 10 °C or 23 °C during 21 h. Oocytes from large follicles (3–7 mm) were classified into BCB+ and BCB- after exposure to BCB (26 μM) for 90 min; then cultured in Medium 199. Oocyte quality was assessed by blastocyst rates after PA in Potassium supplemented simplex optimized medium (KSOM) with 5 % CO2 (condition 1) or in Global Total (GT) medium with 5 % CO2, 5 % O2, and 90 % N2 (condition 2). Mann-Whitney U non-parametric statistics test was used to compare the BCB status and the control group (BCB-free). BCB-free and BCB+ oocytes had higher maturated oocyte than BCB- oocytes after 48 h of IVM (34.7 %, 29.1 % vs.11.7 %); and after 36 h of IVM, BCB-free vs. BCB+ oocytes (55.1 % vs. 51.5 %). In addition, BCB-free group had a higher cleavage rate. Under condition 2, only blastocysts were obtained in both BCB-free (10.6 %) and BCB+ (6.3 %). Therefore, we conclude BCB is a tool for identifying the developmental competence after PA in oocytes BCB stained previously to IVM. Moreover, our findings demonstrate that BCB status does not affect the developmental competence of alpaca oocytes. However, transport temperature, IVM time, culture medium, and culture conditions significantly impact their developmental competence.