Effects of Activation Timing on the Fertilization Rate and Early Embryo Development in Porcine ROSI Procedure

Abstract

Purpose : This study was undertaken to evaluate the optimal exposure time for nuclear envelope breakdown (NEBD) after injection of a round spermatid and to investigate the effect of oocyte activation timing on the fertilization rate and early embryo development in porcine round spermatid injection procedure. Methods : Injected oocytes were fixed at 0.5, 1, 2, 3, and 4 h before activation, and NEBD state was examined. The three groups of oocytes were activated before and after the injection of spermatid using a single direct current pulse (100 V/mm, 50 µs): group 1) at 2 h before the injection (pre), group 2) within 0.5 h after the injection (immediate), and group 3) at 2 h after the injection (post). Activated oocytes were cultured and pronucleus formation and blastocyst development was evaluated at 15-18 h and 7-8 days after the injection, respectively. Results : The proportion of oocytes with NEBD significantly increased in the groups with over 2 h of exposure time ( p < 0.05) and oocyte with premature chromosome condensation began to appear 3 h after the injection. Normal fertilization and development rate to the blastocyst stage were significantly higher in the post group than in those of the pre or immediate group ( p < 0.05). Conclusion : The optimal exposure time for NEBD is 2 h after the injection, and activation in 2 h after round spermatid injection improved the normal fertilization and early embryo development rate.

Molecular Cloning and Characterization of Phospholipase C Zeta in Equine Sperm and Testis Reveals Species-Specific Differences in Expression of Catalytically Active Protein1

“…For instance, in the mouse, rabbit, rat, and pig, round spermatids do not yet express PLCZ [15,[34][35][36][37][38][39]47], because the expression of the protein is first evident at the elongated spermatid stage [15,34,36]. Whereas in the mouse normal offspring have been produced by the injection of these round cells, an additional activation mechanism is necessary to initiate [Ca 2þ ] i rises and, thus, the embryonic development program [34,36].…”

Intracytoplasmic sperm injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation

“…Intracytoplasmic sperm injection has been used widely for the production of offspring in species ranging from the mouse (Kimura and Yanagimachi 1995) to the human (Devroey and Van Steirteghem 2004). However, success with this technique has been very limited in large domestic livestock species, namely the bovine, porcine and equine (Suttner et al 2000;Bedford et al 2003;Choi et al 2004). With the exception of the birth of several calves (Goto et al 1990;Horiuchi et al 2002;Wei and Fukui 2002;Galli et al 2003), conventional ICSI techniques have failed to produce physiological rates of embryonic and fetal development in the absence of an exogenous activation stimulus, such as calcium ionophore and an inhibitor of M-phase cyclins (or their respective cyclin-dependent kinases; Rho et al 1998;Chung et al 2000;Suttner et al 2000).…”

Characterization of Equine Phospholipase C Zeta: A Review and Preliminary Results on Expression Defects in Subfertile Stallions

“…For instance, PLCZ mRNA has been detected as early as the round spermatid stage in mouse testis [15]; however, in many species, including the mouse, as well as the rabbit, rat, and pig, it appears that either mRNA or protein expression is not yet observed at this stage [18,[43][44][45][46][47][48][49], being first evident in elongated spermatids [18,43,45]. Therefore, in these species, generation of offspring through injection of round spermatids would require additional oocyte activation methods to initiate [Ca 2þ ] i increases and thus the embryonic developmental program, as shown in the mouse [45].…”