Polyspermy remains the primary limiting factor for successful porcine embryo production in vitro. It has recently been demonstrated that fertilization of porcine oocytes performed in a microfluidic device led to a significant reduction in polyspermy over conventional IVF, while retaining overall penetration rates. The purpose of this study is to determine the mechanism by which the microchannel environment yields increased rates of monospermic fertilization compared to conventional methods of porcine IVF. OBJECTIVES: It has recently been demonstrated that fertilization of porcine oocytes performed in a microfluidic device led to a significant reduction in polyspermy over conventional IVF, while retaining overall penetration rates. While it is theorized that this is a result of microchannel geometries and flow characteristics, the precise mechanism and critical attributes of the microchannel remain unknown. The goal of this project is to characterize the behavior of sperm and the nature of sperm/oocyte interactions in the microchannel environment. Phase I will involve the adaptation of micro particle image velocimetry to enable tracking of sperm behavior with respect to microchannel geometries and flow characteristics, throughout the 6 hr fertilization incubation. The following questions will be addressed: 1) How many sperm actually flow into the channel, and how does the concentration of sperm in the channel vary over time? 2) At what flow velocity do sperm gain control over their own position, as opposed to being swept along with the flow? 3) Once sperm gain control of their position, where do they choose to reside? i.e. are they evenly distributed throughout the channel or do they tend to cluster near certain geometric features, reside more at the top or bottom of the channel, etc. Does the distribution change after flow subsides? 4) What is the ratio of motile to non-motile sperm at various locations and time points? 5) Is there a specific migration of sperm toward the oocytes after flow has subsided? Can this be explained by geometry alone or is there evidence of chemo-attraction? A final set of experiments will monitor oocytes to determine the conditions of flow and sperm distribution in the microchannel during which successful binding and fertilization most often occurs. The information gathered will facilitate the development of highly efficient fertilization devices in the future. APPROACH: The proposed studies will be conducted using fertilization protocols and microchannel devices identical to those employed by Clark et al. 2005, which proved to yield a dramatic reduction in polyspermy. Oocytes are loaded into the microchannels and come to rest at a constriction within the channel, which is too small for them to pass. Sperm, which can easily fit through the constriction, are added to the inlet reservoir of the channel and are carried past the oocytes by gravity driven flow, due to a slight pressure head in the inlet reservoir. Micro Particle Image Velocimetry (micro-PIV) will be used to characterize the media flow, as well as to track sperm behavior throughout the six-hour incubation. In its purest form, micro-PIV is a method in which fluid seeded with fluorescent nano-particles is introduced into a microchannel. Images are obtained using an epi-fluorescent microscope with pulsed laser illumination. By comparing images taken at short time intervals, particle by particle comparisons (or more commonly correlations between groups of particles) can be made and velocity vectors calculated. By taking advantage of the limited depth of field of the microscope at high magnification 3D reconstructions of the flow field are possible. With adaptations, this system can be used to track sperm motion in addition to the fluorescent nano-particles. This will require optimizing the lighting and image processing to eliminate the need for dyes, and optimizing image capture algorithms to produce the most accurate vector fields. The microscope will also be fitted with a special environmental chamber to maintain a 39C, humidified, five percent CO2 environment throughout the six-hour observation time
