217 KINETICS OF SPERM PENETRATION IS CORRELATED WITH IN VITRO FERTILITY OF BUFFALO (BUBALUS BUBALIS) BULLS

It was previously demonstrated that the kinetics of early cleavage could be used to discriminate between bovine bulls with high and low field fertility (Ward F et al. 2001 Mol. Reprod. Dev. 60, 47–55). Marked differences exist in the kinetics of sperm penetration between bulls, and this may be a useful predictor of field fertility in cattle (Ward F et al. 2002 Theriogenology 57, 2105–2117). It is well known that the ability to fertilize oocytes in vitro and to sustain embryo development varies significantly among buffalo bulls. Therefore, the aim of this work was to evaluate whether the speed of oocyte penetration after IVF was correlated with the blastocyst rates obtainable with different bulls in buffalo species. In Experiment 1, in vitro-matured buffalo oocytes were co-incubated with MitoTracker-labeled spermatozoa (Ward F et al. 2002 Theriogenology 57, 2105–2117) from 6 different bulls, over 2 replicates. Oocytes were subsequently fixed every 3 h (up to 18 h) postinsemination (pi). At each time point, oocytes were denuded, dezoned, fixed in ethanol overnight, and stained with 4′,6-diamidino-2-phenylindole for nuclei examination under a fluorescence microscope. In Experiment 2, in vitro-matured oocytes were fertilized with sperm from the same 6 bulls and were cultured to the blastocyst stage, over 4 replicates. Bulls were tested, collectively, on each batch of ovaries in both experiments. Differences in the percentages of monospermic penetration among bulls were analyzed by chi-square test. Correlation and multiple regression analyses were also carried out between the speed of penetration and blastocyst yields. Marked differences in the kinetics of sperm penetration were found among buffalo bulls, as shown in Table 1. Interestingly, a correlation was found between the blastocyst rate and the percentage of oocytes penetrated at 6 h (r = 0.71; P < 0.01), at 9 h (r = 0.65; P < 0.05), at 12 h (r = 0.77; P < 0.01), and at 18 h pi (r = 0.59; P < 0.05). Regression analysis showed that the optimal time of penetration for predicting the blastocyst rate was 12 h pi (R2 = 0.6). In conclusion, the kinetics of sperm penetration may be a useful marker to predict the in vitro-fertilizing ability of buffalo bulls. The great variability in the speed of oocyte penetration suggests inserting this assessment in the preliminary screening of bulls before their utilization in IVF programs. This may be helpful in selecting high-fertility bulls and identifying the optimal gamete co-incubation times for each bull used. Table 1.Percentage of oocytes penetrated at each time point (hpi, h postinsemination) by different bulls1