**3. Results**

#### **3.1 Dose-response effect of Vn on sperm penetration after bovine IVF**

Compared to the control (0 nM Vn), sperm penetration significantly improved when supplementing 100 nM Vn during IVF (P<0.05; Fig.1). Sperm penetration was significantly inhibited in a concentration-dependent manner starting from 500 nM Vn. This suggests that at higher concentrations, the inhibiting effect of Vn dominates the beneficial effect observed at lower concentrations. In order to determine the mechanism underlying the inhibitory effect of Vn on sperm penetration, subsequent experiments were performed using 500 nM Vn.

#### **3.2 Effect of Vn on sperm penetration of the cumulus oophorus**

When 500 nM Vn was supplemented to the fertilization medium, sperm penetration percentages and fertilization percentages in both CE and CD oocytes decreased significantly compared to the respective control group (Table 1). The difference in reduction of sperm

Tyrode balanced salt solution (control), modified Tyrode balanced salt solution supplemented with 200 nM Vn (100 nM Vn) and modified Tyrode balanced salt solution supplemented with 1 µM Vn (500 nM Vn). Three aliquots from each sperm fraction were incubated (39°C; 5% CO2), and at three different time points of incubation (1 h, 3 h and 6 h, respectively) one aliquot per fraction was evaluated for membrane integrity and total versus

Membrane integrity was evaluated using a fluorescent SYBR14-Propidium Iodide (PI) staining technique (L7011; Molecular Probes, Leiden, The Netherlands). A stock solution of 1 mmol L-1 SYBR14 reagent was diluted (1:50) in HEPES-TALP, stored frozen at –20°C and thawed just before use. From each sperm aliquot, 100 l was used and 1 L SYBR14 was added. After 5 min of incubation (at 37°C), 1 L PI was added prior to another 5 min incubation (at 37°C). Per aliquot 200 spermatozoa were examined using a Leica DMR fluorescence microscope. Three populations of sperm cells were identified: living (membrane intact; stained green), dead (membrane damaged; stained red), and moribund (double stained; green-orange) spermatozoa. The moribund sperm cells were considered to

Total and progressive motility were determined by means of computer-assisted sperm

Differences in fertilization and penetration percentages, and differences in number of Vnpositive cells were analyzed by means of binary logistic regression (including the effect of replicate). To evaluate the differences in mean number of spermatozoa bound to the ZP, the non parametric Kruskal Wallis test was applied, since the concerning variable was not normally distributed. Differences in mean number of sperm cells binding the oolemma were analyzed using ANOVA. Differences in membrane integrity and (total and progressive) sperm motility were evaluated using repeated measures analysis of variance. Hypothesis testing was performed using a significance level of 5% (2-sided test) and results were cited

Compared to the control (0 nM Vn), sperm penetration significantly improved when supplementing 100 nM Vn during IVF (P<0.05; Fig.1). Sperm penetration was significantly inhibited in a concentration-dependent manner starting from 500 nM Vn. This suggests that at higher concentrations, the inhibiting effect of Vn dominates the beneficial effect observed at lower concentrations. In order to determine the mechanism underlying the inhibitory effect of

When 500 nM Vn was supplemented to the fertilization medium, sperm penetration percentages and fertilization percentages in both CE and CD oocytes decreased significantly compared to the respective control group (Table 1). The difference in reduction of sperm

Vn on sperm penetration, subsequent experiments were performed using 500 nM Vn.

progressive sperm motility.

be part of the dead sperm population.

**2.12 Statistical analyses** 

as mean ± S.E.M. (SPSS 15.0).

**3. Results** 

analysis (Hamilton-Thorne CEROS 12.3) (Tanghe *et al.* 2004*a*).

**3.1 Dose-response effect of Vn on sperm penetration after bovine IVF** 

**3.2 Effect of Vn on sperm penetration of the cumulus oophorus** 

penetration was not statistically significant when comparing the CE and the CD groups (P=0.106). Nevertheless, considering the small sample size (n=6), the mean difference of 30.2% in inhibition of penetration between cumulus-enclosed and cumulus-denuded groups suggests a relevant effect of cumulus denudation.

Fig. 1. Dose-response effect of vitronectin (Vn) on sperm penetration after bovine IVF. Data represent mean ± SEM. \*Values significantly different from control with 0 nM Vn (P < 0.05).


Table 1. Fertilization, polyspermy and penetration percentages of cumulus-denuded (CD) and cumulus-enclosed (CE) oocytes inseminated in standard fertilization medium and in fertilization medium supplemented with 500 nM of vitronectin (Vn). a,b Values with a different superscript in the same column within the CD and the CE groups differ significantly (P<0.05).
