Bovine non-competent oocytes (BCB–) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development

Abstract

SummaryCompetent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus–oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 – 60%) and embryonic development rates to the blastocyst stage (10/48 – 21%) as the results obtained with the BCB control group oocytes (45/77 – 58% and 08/45 – 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB– oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB– (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB–; and (4) BCB+ matured in same IVM medium drop as (5) BCB– at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage rate (48 h after IVF), only group 3 (102/229 – 44%) differed (P < 0.05) from the other groups [1 (145/241 – 60%); 2 (150/225 – 67%); 4 (201/318 – 63%) and 5 (21/33 – 63%)]. On day 7, the embryos from group 2 (BCB+) achieved the highest blastocyst rate (46/150 – 31%) (P < 0.05) when compared with the embryo development capacity of the other experimental groups (1: 31/145 – 21%; group 3: 17/102 – 17%; group 4: 46/201 – 23%; and group 5: 2/21 – 10%). In conclusion, submitting BCB+ oocytes that were separated from BCB– oocytes to IVM increases the rate of embryonic development to the blastocyst stage when compared to the control group, BCB– oocyte group, BCB+ paracrine group and BCB– paracrine group. The presence of non-competent oocytes during IVM, even in low proportion (1:10), reduces the capacity of competent oocytes to undergo embryo development and achieve blastocyst stage during IVC.