Effects of ultrashort gamete co-incubation time on porcine in vitro fertilization.
A reduction in co-incubation time has been suggested as an alternative method to reduce polyspermic fertilization. The aim of this study was to evaluate the effect of short periods of gamete co-incubation during pig in vitro fertilization. A total of 2,833 in vitro matured oocytes were inseminated with thawed spermatozoa and coincubated for 0.25, 1, 2, 3, 7, 10 min and 6h. The oocytes from the 0.25-10 min groups were washed three times in modified Tris-buffered medium (mTBM) medium to remove spermatozoa not bound to the zona and transferred to the same medium (containing no spermatozoa) until 6h of co-incubation time were completed. After 6h, presumptive zygotes from each group were cultured in NCSU-23 medium for 12-15 h to assess fertilization parameters. After each period of co-incubation, 45-50 oocytes from each group were stained with Hoechst-33342 and the number of spermatozoa bound to the zona was counted. Although the number of zona bound spermatozoa increased (p<0.05) with the co-incubation time, no increase was observed in penetration rates among groups from 2 min to 6h of co-incubation time (ranging from 53.5+/-2.8 to 61.3+/-2.6%). Similarly, the efficiency of fertilization reached a maximum for the 2 min of co-incubation group with values ranging between 32.3+/-2.4 and 41.9+/-2.5%. The reduction of co-incubation time did not affect the monospermy rate (range: 71.3+/-3.4-80.2+/-3.8%) and the mean number of spermatozoa/oocyte (range: 1.2+/-0.4-1.4+/-0.5). These results show that, under our in vitro conditions, high penetration rate can be obtained with co-incubation times as short as 2 min, although monospermy could not be improved using this strategy.
Almiñana
C
C
Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain.
Gil
M A
MA
Cuello
C
C
Parrilla
I
I
Roca
J
J
Vazquez
J M
JM
Martinez
E A
EA
D000818:Animals; D004526:Efficiency; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D008297:Male; D011247:Pregnancy; D018873:Pregnancy Rate; D011270:Pregnancy, Animal; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013552:Swine / Q000502:physiology*; D013997:Time Factors