The effect of sperm preparation and co-incubation time on in vitro fertilization of bos indicus oocytes

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm–oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18 h. After co-incubation oocytes were transferred to the culture medium and culture for 44 h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm–oocyte co-incubation time was observed (P>0.05). However, sperm–oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6 h (63.3 and 54.4%) and 12 h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18 h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12 h, and the prolongation of that time for up to 18 h had no detrimental effect on fertilization.