173 Assessment of the first polar body quality and viability in bovine

Abstract

In female gametes, after the first asymmetric meiotic division, a mature oocyte in metaphase II and a first polar body (PB1) are generated. The PB1 contains one of each pair of homologous chromosomes present in the mature oocyte and its DNA can be used for preconception genetic diagnosis. The PB1 degenerates shortly after extrusion, possibly due to an apoptotic process; however, it has not yet been elucidated in bovine. Therefore, the objective of this study was to evaluate PB1 morphology changes, plasma membrane integrity, and the presence of DNA fragmentation during in vitro maturation (IVM). To this aim, cumulus-oocyte complexes collected from slaughterhouse ovaries were cultured in maturation medium in different groups according to IVM time: 16, 18, 20, 22, 24, and 26 h. The PB1 were classified into 5 categories according to their morphology: grade (G)1, round PB1 with intact smooth membrane; G2, round or ovoid PB1 with intact membrane; G3, broken PB1 with a small PB1 fragment; G4, broken PB1 with a big PB1 fragment; and G5, completely damaged PB1. Grades 1 and 2 were considered good quality. Plasma membrane integrity was assessed by propidium iodide (PI) DNA staining; PI is a fluorescent intercalating agent that cannot cross the membrane of live cells. The presence of DNA fragmentation was detected at 16, 22, and 26 h by TUNEL assay. Data were analysed by two-way ANOVA and Bonferroni post hoc test using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA) and differences were considered significant at P < 0.05. Our results (mean % ± s.e.m.) showed that significantly more oocytes assessed at 18, 20, 22, and 24 h after onset of IVM presented high-quality (G1) PB1 (18 h: 61.5 ± 13.4%, 20 h: 73.5 ± 9.6%, 22 h: 61.0 ± 9.5%, 24 h: 60 ± 5.1%), compared with those assessed at 16 and 26 h (43.0 ± 4.7%, 22.3 ± 3.4%, respectively). The percentage of G2 PB1 did not change throughout the period studied (16 h: 34.0 ± 13.5%, 18 h: 29.9 ± 14.1%, 20 h: 22.0 ± 7.1%, 22 h: 26.5 ± 4.2%, 24 h: 23.3 ± 4.9%, 26 h: 21.33 ± 9.9%), but was significantly lower than that of G1 PB1 at 20, 22, and 24 h. The proportion of damaged (G5) PB1 started to increase at 24 h (14.3 ± 8.6%), being highest at 26 h (30.0 ± 10.5%), in parallel with positive PI staining (P < 0.05). Moreover, there was a significant increase of PB1 with DNA fragmentation at 26 h (82.0 ± 18.0%) compared with 16 h (13.9 ± 9.0%) and 22 h (2.5 ± 2.5%). Altogether, these findings demonstrate that PB1 remains stable and of good quality between 18 and 24 h; however, after this time, plasma membrane integrity is compromised and the DNA is fragmented, suggesting the occurrence of an apoptotic process. Our results could be helpful to determine the optimal time for using PB1 as a potential donor of genetic material.