Kinetics of sperm penetration in buffalo (Bubalus bubalis) species: effect of bull

The aim of this work was to evaluate whether the kinetics of sperm penetration varies among buffalo bulls. To assess penetration, in vitro matured buffalo oocytes were inseminated with MitoTracker-labelled spermatozoa (Ward, 2002, Theriogenology, 2105-2117) from 4 different bulls (n = 248, 254, 269 and 217 respectively with bulls A, B, C, D, over 3 replicates). Oocytes were subsequently fixed every 3 h (up to 18 h) post-insemination (pi). At each time point, oocytes were denuded, dezoned, fixed in ethanol overnight and stained with DAPI for nuclei examination under fluorescence microscope. Differences in the percentages of monospermic penetration among bulls were analyzed by Chi square test. Bull A showed higher (P<0.01) penetration than bulls B, C and D at 3 h pi (67, 12, 6 and 0 %), at 6 h pi (74, 26, 26 and 3 %), at 9 h pi (79, 36, 49 and 8 %) and at 12 h pi (63, 49, 54 and 16 %). At 15 h pi bull C reached the same penetration rate of bull A (69 and 71 % vs 46 and 24 % respectively for bulls A, C, B and D; P<0.01). At 18 h pi bulls A, B and C reached similar percentages of penetration (60, 76, 72 vs 26 % respectively for bulls A, B, C and D; P<0.01). Bull D exhibited the lowest penetration rate at all time points studied. In conclusion, marked differences in the kinetics of sperm penetration exist among buffalo bulls, suggesting a possible correlation with fertility that needs to be investigated. Furthermore, this finding suggests to vary the gametes co-incubation time in relation to the bull used for in vitro fertilization.