215 APPLICATION OFA MICROFLUIDIC SPERM SORTER TO THE IN VITRO FERTILIZATION OF PORCINE OOCYTES

Abstract

In porcine IVF, polyspermy is a persistent obstacle to the efficient production of normal embryos in vitro. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) has been developed to isolate motile human spermatozoa (from diluted semen by 2 laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. In addition, we have succeeded in reducing the incidence of polyspermic penetration by a transient coincubation of oocytes with spermatozoa in the presence of caffeine, followed by culture of the oocytes in the absence of caffeine (2004 Reproduction 128, 789–800). If porcine oocytes could be cocultured with motile spermatozoa in the chamber of the MFSS, the gradual accumulation of motile spermatozoa might further improve the efficiency of production of monospermic fertilized porcine embryos. The current study was undertaken to apply the MFSS technology to porcine IVF. The sperm-rich fraction from 3 Berkshire boars was diluted at 1 × 10⁸ cells mL^–1 with modified Modena containing 20% seminal fluid, cooled to 15°C for 4 h, and kept at the same temperature until use within 2 days. Stored, diluted semen with greater than 80% viability was flowed with Tyrode lactate-HEPES-polyvinyl alcohol medium for 5 or 10 min at room temperature. The concentration and viability of spermatozoa mixed in the flowed medium were determined. In the next experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine for 5 min at room temperature. Before flowing, porcine IVM oocytes were put into the chamber of the MFSS, where motile spermatozoa will accumulate with modified TCM-199 containing 5 mm caffeine. After flowing for 5 min, those oocytes were transferred into a 500-µL droplet of caffeine-free modified TCM-199, and culture was continued for 8 h. Statistical analyses were carried out by ANOVA and Fisher's PLSD post hoc test. After flowing for 5 min, the concentration of spermatozoa recollected from the chamber was 5.7 × 10⁵ cells mL^–1. The viability of recollected spermatozoa was significantly higher than the original flowed semen. However, when spermatozoa were flowed for 10 min, the viability of recollected spermatozoa was not different from the original flowed semen, whereas the concentration of recollected spermatozoa was 9.7 × 10⁵ cells mL^–1. When IVM oocytes were cocultured with spermatozoa gradually accumulated in the chamber of MFSS for 5 min, 35% (22 to 48%) of oocytes were penetrated, and 95% (90 to 100%) of the penetrated oocytes were monospermic. These observations demonstrate that the MFSS can separate penetrable boar spermatozoa with a high viability and sufficient concentration for IVF. Furthermore, the current data suggest the possibility of improving the efficiency of monospermic fertilization of porcine IVM oocytes by a transient coculture with MFSS.