Viscoelastic Modifiers and Hyperactivating Agents as Potential Selectors of Superior Sperm for in Vitro Fertilization

Efficiency of bovine in vitro fertilization is lower than that of in vivo fertilization (IVF) potentially due to less stringent selection of sperm. Sperm hyperactivation is essential for fertilization and in aqueous media, hyperactivated sperm swim in tight circles but in viscoelastic media, such as medium containing long-chain polyacrylamide (PA) and uterine tubal fluid, hyperactivated sperm move progressively whereas non-hyperactivated sperm are unable to traverse such media. Our objectives were to: 1) identify hyperactivating conditions that improve sperm penetration of viscoelastic media; and 2) determine whether in vitro fertilization (IVF) in viscoelastic media improves cleavage rate. The hypotheses were that: 1) sperm hyperactivating agents such as caffeine, procaine, and 4-aminopyridine (4-AP) or in combinations would improve sperm motility in viscoelastic media; and 2) performing IVF in viscoelastic media would improve fertilization rates. Experiment 1 tested the effects of 1 % and 1.5 % PA and hyperactivating agents compared to control (aqueous media) on sperm movement parameters measured by CASA (CEROS, Hamilton Thorne, Beverly, MA); three bulls were tested and each bull was tested in three replicates. Outcome measures were: % total motile, % progressively motile, velocity average path (VAP), velocity curvilinear (VCL), velocity straight line (VSL), straightness (STR) and linearity (LIN). Linear regression (JMP Pro 11, SAS Institute, Cary, NC) was used to determine effects of treatments on CASA outcomes controlling for bull effects. In PA, VAP, VCL and VSL were decreased (P < 0.05) compared with control but STR and LIN were increased (P < 0.05). Total and progressive motility were lower (P < 0.005) in 1.5% but not in 1% PA compared with control media, but linearity and straightness were increased in both concentrations of PA (P < 0.01). Hyperactivating agents individually were not effective but when used in combinations had additive effects and (P < 0.05) increased hyperactivation. In particular, 4-AP and caffeine were effective to hyperactivate sperm in viscoelastic media. In Experiment 2, an IVF trial was performed to determine if fertilization in viscoelastic media (1.5% PA) or supplemented with 4-AP in addition to caffeine or PA plus 4-AP improved cleavage rates. Bovine embryos were produced from slaughterhouse ovaries in four replicates of IVF experiments (n = 823 oocytes). Mixed logistic regression (STATA 10.1, College Station, TX) with replicate treated as a random variable revealed that both PA and 4-AP treatments decreased cleavage rates (P < 0.001) although there were no additive effects of the two factors (caffeine + 4-AP). In conclusion, velocity parameters of sperm were decreased in viscoelastic media but STR and LIN were increased. Both PA and 4-AP decreased cleavage rates in IVF. This may be due to either direct damage to sperm or oocyte during incubation by these factors.