title: Addition of D-penicillamine, hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro.
abstract: The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10(6) cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10(6) cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10(6) cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10(6) cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.
authors.affiliation: Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors.forename: Sung-Sik
authors.initials: SS
authors.lastname: Kang
authors.forename: Keisuke
authors.initials: K
authors.lastname: Koyama
authors.forename: Weiping
authors.initials: W
authors.lastname: Huang
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Yang
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Yanagawa
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Masashi
authors.initials: M
authors.lastname: Nagano
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000187:drug effects*; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D004837:Epinephrine / Q000008:administration & dosage*; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D008297:Male; D009865:Oocytes / Q000378:metabolism; D010396:Penicillamine / Q000008:administration & dosage*; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D013654:Taurine / Q000008:administration & dosage / Q000031:analogs & derivatives*
chemical_list: D013654:Taurine; C003949:hypotaurine; D000242:Cyclic AMP; D010396:Penicillamine; D004837:Epinephrine