Damage-free Sorting of Sperm
Sperm cells are known to be extremely sensitive to SICS, and a large number of sperm that are sorted using a conventional sorter are not suitable for use in in vitro fertilization (IVF). The effect of sperm sorting on On-chip Sort was evaluated using parameters such as motility, fertility and developmental ability. The overall procedure of the experiments is presented on Fig. 1. Oocytes and sperm were retrieved from C57BL/6L mice and sperm were pre-incubated in BSA-free Toyoda Yokoyama Hosi medium. Pre-incubated sperm was sorted using On-chip Sort with calcium-enhanced human tubal fluid (mHTF) as sheath fluid. IVF was performed on a dish with the collected sperm and retrieved oocytes in mHTF. In order to determine the developmental ability of the cultured two-cell embryos produced by IVF using collected sperm, embryo were transferred to Jcl:ICR mice.
Fig. 1. Overall experimental procedure.
Sperm sorting on On-chip Sort was performed based on signals from forward scattered light (FSC) and side scattered light (SSC). The sorted sperm had lower motility than unsorted sperm (Fig. 2a), but the fertilization rate showed no significant difference between unsorted and sorted sperm (Fig. 2b). The sorted sperm were fertilized with oocytes, and the fertilized eggs developed normally up to the blastocyst stage in in vitro culture, which was comparable to those that were unsorted (Fig. 2c). Normal pups were obtained after embryo transfer, and birth rate was similar for both that used unsorted and sorted sperm (Fig. 2d). On-chip Sort has shown the mouse sperm isolation capability while maintaining normal fertilization and developmental ability.
Fig. 2. (a) Motility of sperm with and without sorting. (b) Fertilization rate of sperm with and without sorting. (c) Developmental rate of sperm with and without sorting at several developmental stages. (d) Birth rate of live pup derived from unsorted and sorted sperm.