Effect of sperm preparation method on in vitro fertilization in pigs.
This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.
Matás
C
C
Department of Physiology (Veterinary Physiology), Faculty of Veterinary Science, University of Murcia, 30071-Murcia, Spain. cmatas@um.es
Coy
P
P
Romar
R
R
Marco
M
M
Gadea
J
J
Ruiz
S
S
D020101:Acrosome Reaction; D000704:Analysis of Variance; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D003470:Culture Media; D005314:Embryonic and Fetal Development; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D008297:Male; D011205:Povidone; D012822:Silicon Dioxide; D013048:Specimen Handling / Q000379:methods*; D013094:Spermatozoa; D013552:Swine; D015053:Zygote
D003470:Culture Media; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone