103 Quality and fertilization of frozen–thawed porcine spermatozoa separated using migration sedimentation

A commercially available device (MIGLIS, Menicon Life Science) for sperm preparation based on the migration sedimentation of spermatozoa can be used to settle motile sperm to the bottom of a tube without centrifugation. The MIGLIS device comprises of a small conical cup (central tube) built into an outer container, in which only motile spermatozoa migrates from the deposition (outer) area into the central (inner) tubule. Based on this selection mechanism, the present study was conducted to evaluate the quality and fertilization of frozen–thawed porcine spermatozoa that migrated to the inner region compared with those that remained in the outer region. In experiment 1, to determine the adequate concentration of caffeine for separation, frozen–thawed spermatozoa were swum in a MIGLIS device containing tissue culture medium (TCM) 199 with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM). In experiment 2, to determine the appropriate incubation time for frozen–thawed spermatozoa in MIGLIS, the spermatozoa were swum in TCM 199 supplemented with 2.5 mM caffeine for 5, 10, 15, and 20 min. After incubation with caffeine, the sperm motility, viability, plasma membrane integrity, and acrosomal integrity were assessed. In experiment 3, to evaluate the fertilization and embryo development of oocytes fertilized with frozen–thawed spermatozoa separated into two regions (outer and inner), in vitro-matured oocytes were fertilized with spermatozoa from two boars and then cultured in vitro for 7 days. Data were evaluated using analysis of variance (ANOVA), followed by Fisher’s protected least significant difference test using STATVIEW. Sperm recovery rates in the inner region were significantly lower than those in the outer region (16.6% vs 63.8%, P < 0.05), but the motility of spermatozoa that migrated to the inner region was higher than that of spermatozoa that remained in the outer region, irrespective of caffeine concentration and incubation time. Supplementation with 2.5 mM caffeine and incubation for 10 min resulted in higher-quality spermatozoa that migrated to the inner region. Furthermore, in the spermatozoa from the two boars, the rates of total fertilization and blastocyst formation of spermatozoa that migrated to the inner region were significantly higher (P < 0.05) than those of spermatozoa that remained in the outer region (total fertilization, 75.7–81.7% vs 54.5–58.0%; and blastocyst formation, 8.0–13.3% vs 3.0–5.9%, respectively, in four replicates). Therefore, the MIGLIS device may be useful for separating high-quality spermatozoa and fertilizing oocytes to improve IVF results.