(59) Canthaxanthin Reduces Polyspermy During In Vitro Fertilization in Pigs

Jinsung Lee¹, Junchul David Yoon², Lillian Miller², Jaehwan Kim¹, Bethany Redel³ and Kiho Lee¹^,²

1. Division of Animal Science, College of Agriculture Food and Natural Resources, University of Missouri, MO, USA

2. National Swine Resource and Research Center, Columbia, MO, USA

3. United States Department of Agriculture – Agriculture Research Service, Plant Genetics Research Unit, Columbia, MO, USA

Abstract Text:

One of the major obstacles to employ in vitro fertilization (IVF) technology in pigs is polyspermy. Often more than 50% of fertilized oocytes present polyspermy and embryo development can be stalled if subsequent embryos become aneuploidy. During fertilization, initial mechanism to block to polyspermy occurs through membrane depolarization shortly after sperm-oocyte fusion triggered by a rise in intracellular calcium concentrations. The TMEM16A, a calcium-activated chloride channel, facilitates this depolarization by allowing Cl^- efflux across the plasma membrane and the rapid electrical changes prevents additional sperm entry. In this study, we utilized Canthaxanthin (CX), a pharmacological activator of TMEM16A, to suppress the frequency of polyspermy during porcine IVF. First, porcine oocytes were collected from ovaries sourced from a local abattoir and In vitro maturation (IVM) was performed using M199 medium, supplemented with epidermal growth factor (EGF), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and FLI (FGF2, LIF, IGF1). After IVM, oocytes reached MII stage were used for IVF. During co-incubation of gametes for five hours, a series of CX (0 (control), 1, 5, 10, and 25 µM) was supplemented to IVF medium. After IVF, fertilized oocytes were then cultured in Pig Zygotic medium. To assess the frequency of polyspermy, pronuclear (PN) formation was visualized at 10 hours after IVF. The differences were compared using Chi-square test and P< 0.05 was considered significant. Across four independent trials, the 10µM CX group presented a significantly lower percentage of embryos carrying more than two pronuclei (P < 0.01); the average of the multiple PNs (2+) was 53.4% in the control and 31.6% in the 10 µM group. Percentage of embryos carrying 2PN was higher in 10µM CX group (48.5%) compared to control (31.6%) and the percentage of unfertilized oocytes was not different among groups. These findings demonstrate that 10µM CX effectively protect porcine oocytes from polyspermy and improves normal fertilization rates in porcine IVF. Future studies will focus on identifying mechanistic actions of CX and monitor developmental potential of embryos exposed to CX.

Keywords: Canthaxanthin, in vitro fertilization, swine embryo, pronuclei