Previous studies have related spermatozoa with increased percentages of PCDs to lower motility in humans [25
]. A similar connection was found in the present study, as seen by the significant (P < 0.001) negative correlation between motility and PCDs. In young beef bulls, zero to six weeks after puberty, the percentage of progressively motile spermatozoa increased rapidly, while the percentage of spermatozoa with proximal cytoplasmic droplets was correspondingly dramatically reduced [26
]. High percentages of ejaculated spermatozoa with retained cytoplasmic droplets are associated with infertility in adult bulls [3
]. A recent review indicated that elimination of the droplets during ejaculation may be prognostic for fertility, while their retention may indicate sub- or infertility [27
Morphologically abnormal spermatozoa are more susceptible to oxidative stress. The production of reactive oxygen species (ROS) by spermatozoa with PCDs can induce oxidative stress [28
], resulting in cellular dysfunction by a number of mechanisms, among them membrane lipid peroxidation [1
]. Lipidic peroxidation was observed in situ
in human [29
] and bovine [28
] spermatozoa midpieces, incorporating the mitochondria and any retained excess residual cytoplasm [29
]. The impact of the cryopreservation process on membranes and mitochondria has been intensely studied [31
]. However, there are few studies indicating that semen with specific morphological alterations would be more sensitive to injuries on these compartments [7
Considering these data, the high percentages of spermatozoa with damaged plasma membranes and acrosomes and with low mitochondrial potential, as observed in group 2 for semen samples with high proportions of PCDs, may have been due to higher production of ROS, resulting in oxidative damage. Although differences were observed in the results obtained for acrosome integrity as analysed using the PIA and triple staining techniques, this discrepancy was due to different methodologies; PIA evaluates the percentage of detached acrosomes after STT (37°C/3h) [13
], while the FITC-PSA fluorescent probe binds the glycoconjugates and thus labels the acrosome content and indirectly determines acrosome integrity [37
]. Nevertheless, the correlation in the results indicates that PCDs have an important effect on acrosome integrity, regardless of the evaluation method.
Semen DNA integrity did not differ between group 1 and group 2; however, the results indicated a higher correlation between the percentage of spermatozoa with PCDs and stable DNA, in particular after STT, indicating that chromatin susceptibility to acid denaturation may be greater after incubation at 37°C, as observed in our previous study [38
Considering the discrepancies among IVF results, individual responses in the IVF process can be assumed. In fact, the bull effect in this experiment was not prevented by pooling the semen. Grouping the bulls according to percentages of spermatozoa with PCDs (group 1/control and group 2/high levels of PCD), the rates of cleavage, blastocyst formation and hatched blastocysts did not significantly differ. A high incidence of proximal droplets is indicative of impaired epididymal maturation in young bulls and may severely compromise IVF results because when they reach sexual maturity, proximal droplets decrease and IVF potential increases [3
]. However, the bulls used in the present study were adults and were sexually mature.
Considering the results from IVF of the two bulls with higher PCD levels (bulls P1 = 28.5% PCD and P2 = 40.5% PCD), P2 had a numerically lower performance in IVF, indicating that the higher levels of PCD may have affected the embryo production efficiency of this animal (P2). Thundathil et al. [7
] suggested that morphologically normal sperm co-existing in the semen along with spermatozoa showing proximal droplets were also functionally deficient. These authors used semen with even higher levels of PCDs (45% to 86%) and found low cleavage rates (8% to 15%) and no blastocyst formation. Nevertheless, the results for control bull C2 were similar to those for the high PCD bull, P2, and this result indicates that, despite our hypothesis regarding the influence of PCDs on IVF rates, the results may have been affected by different variables, such as individual bull characteristics. Bull effects on sperm motility [39
], IVF results and embryo development rates have been observed in a number of investigations [19
]. Both fertilising ability and competency for embryonic development have varied significantly among the semen of individual bulls [41
]. Individual semen characteristics were found not to affect cleavage but to affect embryo development [40
Our results emphasise the importance of the genetic background of individual semen donors for successful IVF procedures, as well as careful semen evaluation.