158 The Effect of Follicular Wave Phase at Time of Ovum Pick-Up on Bovine Oocyte Cytoplasmic Maturation and Developmental Competence

Abstract

During oocyte collection, follicular wave phase is unknown, although differences in follicle environment may have dramatic effects on oocyte quality. This project was performed to determine whether oocyte collection during different phases of the follicular wave affects oocyte competence. Oocytes were collected via transvaginal ultrasound guided oocyte aspiration from 18 cows, at 4, 8, and 12 days following dominant follicle removal, representing follicle wave emergence, peak, and atresia, respectively (160, 314, and 273 oocytes, respectively). Once recovered, oocytes were graded and assigned to either being held as immature or matured in vitro for 24 h. Oocytes were then stained in Mitotracker deep red, fixed and stained with an anti-IP₃R1 primary antibody and an Alexa Fluor 488-conjugated secondary antibody, before being stained with DAPI, to identify mitochondria, inositol triphosphate receptor 1 (IP₃R1), and chromatin respectively. Mitochondria were analysed based on cytoplasmic distribution and classified as peripheral (immature), diffuse, central (mature), or sparse. Expression of IP₃R1 was measured as corrected total cell fluorescence in Image J (National Institutes of Health, Bethesda, MD, USA). Staining patterns were analysed using ANOVA. A subset of the matured oocytes was stained with Fluo-3 to measure cytoplasmic calcium levels. These oocytes were then parthenogenetically activated before being imaged again to view changes in calcium levels, and presumptive embryos were cultured for 4 days. Fluo staining was measured as intensity levels (none, slight, moderate, high) and differences in development and stain levels were analysed using the Kruskal-Wallis test. Although mitochondria location was unaffected by collection day, it was significantly affected by maturation status (P = 0.0036). However, oocytes showed incomplete mitochondrial maturation, with mitochondria residing in the diffuse orientation in the majority of oocytes. Expression of IP₃R1 appeared to be more sensitive to treatment. Expression significantly increased as meiosis proceeded (P = 0.0081) and there was a significant difference in expression between oocyte collection days (P = 0.0026). The interaction between collection day and maturation status also had a significant effect (P = 0.048), with mature oocytes showing an increase in IP₃R1 expression, most notable in those collected on Day 4. Oocyte quality had a notable effect on the ability of oocytes to progress through meiosis (P = 0.054) and on mitochondrial location (P = 0.053), with AB oocytes showing better maturation parameters in both respects. Although the day of collection did not affect embryo development, Fluo stain intensity was an indicator of embryo developmental potential (P = 0.053), with oocytes having decreased potential to develop if the initial calcium levels were moderate to high. Results suggest that oocyte collection during wave emergence yields a slight advantage in oocyte quality. Although IP₃R1, necessary for Ca²⁺ spikes during fertilization, indicates competence, high levels of cytoplasmic Ca²⁺ at the time of activation appear to be detrimental to embryo development.