Traditional methods of oocyte selection for in vitro studies have proven inefficient with respect to achieving a level of predictability for competency. In this study, a novel method of oocyte selection was implemented that identified a relationship between oocyte morphological parameters (as defined by a ratio of a shape factor (SF) to average fluorescence intensity (AFI) and AFI, followed by in vitro fertilization (IVF) and in vitro culture (IVC) using the Well of Well (WOW) method to evaluate oocyte competency. Specifically, we used non-cytotoxic fluorescent molecular probes and multiphoton microscopy to non-invasively characterize spatial localization and functional activity of mitochondria, mitochondrial membrane potential (Δψm), and intracellular calcium activity ([Ca2+]i) using rhodamine 123, JC-1 and Fluo-4, AM, respectively in bovine and canine in vitro matured (IVM) oocytes. Comparison of morphological grading with fluorescence intensity yielded similar trends between all grades of oocytes for both species with no visually obvious, distinct characteristic staining that would permit classification of each oocyte as a specific morphological grade. Our studies confirmed that oocyte mitochondria were homogeneously distributed but primarily localized to the peri- and sub-cortical regions of the oocyte at MII stage for both species. Further, heterogeneously polarized mitochondria were localized to the peri- and sub- cortical regions of the oocyte for both species. In bovine oocytes labeled with Fluo-4, AM, levels of [Ca2+]i were either unremarkable, or very low and limited to the peri-cortical areas, just beneath the oolema. For canine MII stage oocytes, levels of [Ca2+]i were within the same range of AFI as bovine. Ranges of fluorescence intensity compatible for optimal embryo development for bovine and optimal fertilization for canine oocytes were 30-300 and 20-35, and 20-30 and 20-25.5 for rhodamine 123 and Fluo-4, AM, respectively. The optimal range for bovine oocytes imaged with JC-1 was 1.25-2.25 and <6 for canine.

Traditional methods of oocyte selection for in vitro studies have proven inefficient with respect to achieving a level of predictability for competency. In this study, a novel method of oocyte selection was implemented that identified a relationship between oocyte morphological parameters (as defined by a ratio of a shape factor (SF) to average fluorescence intensity (AFI) and AFI, followed by in vitro fertilization (IVF) and in vitro culture (IVC) using the Well of Well (WOW) method to evaluate oocyte competency. Specifically, we used non-cytotoxic fluorescent molecular probes and multiphoton microscopy to non-invasively characterize spatial localization and functional activity of mitochondria, mitochondrial membrane potential (Δψm), and intracellular calcium activity ([Ca2+]i) using rhodamine 123, JC-1 and Fluo-4, AM, respectively in bovine and canine in vitro matured (IVM) oocytes. Comparison of morphological grading with fluorescence intensity yielded similar trends between all grades of oocytes for both species with no visually obvious, distinct characteristic staining that would permit classification of each oocyte as a specific morphological grade. Our studies confirmed that oocyte mitochondria were homogeneously distributed but primarily localized to the peri- and sub-cortical regions of the oocyte at MII stage for both species. Further, heterogeneously polarized mitochondria were localized to the peri- and sub- cortical regions of the oocyte for both species. In bovine oocytes labeled with Fluo-4, AM, levels of [Ca2+]i were either unremarkable, or very low and limited to the peri-cortical areas, just beneath the oolema. For canine MII stage oocytes, levels of [Ca2+]i were within the same range of AFI as bovine. Ranges of fluorescence intensity compatible for optimal embryo development for bovine and optimal fertilization for canine oocytes were 30-300 and 20-35, and 20-30 and 20-25.5 for rhodamine 123 and Fluo-4, AM, respectively. The optimal range for bovine oocytes imaged with JC-1 was 1.25-2.25 and <6 for canine.