The use of in vitro fertilization techniques to investigate the fertilizing ability of bovine sperm with proximal cytoplasmic droplets.

The objective of this experiment was to determine the effect of proximal droplets on sperm-oocyte binding, zona penetration, fertilization, and the developmental competence of oocytes fertilized by sperm with proximal droplets (PD) in an in vitro fertilization (IVF) and culture system. Frozen semen from three bulls (PD1, PD2 and PD3) with varying proportions of normal appearing sperm with proximal droplets and semen from a normal control bull (C) were used in this experiment. The mean number of sperm bound to the zona pellucida (26.8 +/- 2.0, n=100; 15.2 +/- 1.1, n=100; 16.2 +/- 1.0, n=100) for bulls PD1, PD2, and PD3, respectively, were significantly lower (P<0.05) than that of the control bull C (47.4+/- 1.9; n=114). No spermatozoa with PD were found bound to the zona pellucida and this finding was consistent among the three bulls. The percentage penetration of zonae for the bulls PD1, PD2 and PD3 (74%, 74/100; 71%, 71/100 and 69%, 69/100, respectively) were not different than that of bull C (72%, 179/245). Similarly, the mean number of sperm penetrating the zona pellucida (1.43+/- 1.2, 1.24 +/- 1.1 and 1.20 +/- 1.1, for bulls PD1, PD2 and PD3, respectively) were not different than that of bull C (1.45 +/- 1.1). However, fertilization rates (8.8%, 8/90; 16.8%, 16/95; and 10.6%, 11/103, for bulls PD1, PD2 and PD3, respectively) were lower (P<0.001) than that of bull C (68.7%; 77/112). Similarly, cleavage rates (5%, 10/200; 8%, 8/100 and 14%, 15/111) for the bulls PD1, PD2 and PD3, respectively, were lower than that of the control bull, C (60.7%; 79/130). Cleaved zygotes resulting from the fertilization of oocytes by apparently normal sperm from bulls with PD did not develop beyond cleavage, whereas, 43.8% (57/130) morulae and 20% (26/130) blastocysts were produced by oocytes fertilized by sperm from bull C. In summary, normal appearing sperm with PD did not bind to the zona pellucida. Apparently normal sperm with out proximal droplets co-existing in the semen along with sperm containing PD were also functionally deficient, resulting in reduced zonae binding and zygotes resulting from insemination with semen with a high percentage of PD did not develop beyond cleavage.