262IN VITRO FERTILIZATION IN MICROFLUIDIC CHANNELS ENHANCES
MONOSPERMIC PENETRATION OF SWINE OOCYTES
Authors: Clark S.G. , Walters E.M. , Beebe D.J. , Wheeler M.B. 
Published: February 2004

In traditional porcine IVF systems, there is a high incidence of polyspermy, a pathological condition which results in aberrant embryonic development. Efforts to improve the in vitro embryo production process in pigs have included modifying the culture medium, the number of spermatozoa inseminated, and the quantity of medium used. Recently, the development of microscale embryo culture devices (microchannels) has opened new avenues for manipulation of the IVF system to improve the efficiency and overall production of porcine embryos by more closely mimicking the function of the oviduct. The volume of medium in the local vicinity of the embryo is smaller (0.125μL) compared to the typical 5–50μL microdrops. Additionally, it is believed that the delivery of sperm cells in the microchannel simulates the flow pattern of spermatozoa past the oocytes similar to that in the oviduct. This study was designed to compare the incidence of polyspermy of pig oocytes fertilized in PDMS-glass microchannels (MC) to conventional microdrop methods (controls). Oocytes were obtained by aspiration of ovarian antral follicles. Fifty oocytes were placed into 500μL of TCM199 medium supplemented with LH, FSH, EGF, cysteine, PVA, and antibiotics. Extended semen was washed with mTBM and re-suspended to 6×105 sperm/mL. The sperm suspension was placed in humidified 5% CO2 in air atmosphere at 39°C for 1h to allow for capacitation. Concurrently, pre-warmed microchannels were filled with 200μL of mTBM and allowed to equilibrate for 1h at 39°C. Cumulus cells were removed from the oocytes using 0.1mgmL−1 hyaluronidase in mTBM. At 44h of maturation, 15 oocytes were placed into a pre-equilibrated 50μL drop of mTBM covered with warm paraffin oil in a petri dish, and placed into the microchannel. Capacitated sperm cells were then added to the oocytes to give a final concentration of 3×105 sperm/mL; the mixture was incubated for 6h. Presumptive zygotes were cultured in 100μL drops of NCSU-23 covered with oil at 39°C in a humidified 5% CO2 in air for 12h. At this time, the zygotes were fixed in 1:3 (v/v) glacial acetic acid in absolute ethanol for 48–72h. Aceto-orcein staining data revealed a higher incidence of monospermic penetration and a lower number of spermatozoa per oocyte in the microchannels as compared to the controls (Table 1). Data from six replicates were arranged in a randomized block design and analyzed by the generalized linear model in SAS. These data support the idea that the microchannel environment reduces the incidence of polyspermy during IVF of porcine oocytes (P&lt;0.05) while maintaining comparable penetration and male pronuclear formation rates. Furthermore, it is possible that the number of sperm present near the oocytes during fertilization is decreased using the microchannel. In conclusion, microfluidic technology has shown the potential to improve in vitro fertilization in swine by an increasing monospermic penetration of oocytes.

Table 1
Effect of IVF system on fertilization parameters