In vitro assessment of fertilization and embryo development with Bovine spermatozoa after scrotal insulation

Fertilization and cleavage of bovine embryos depend not only on maternal involvement, but also on the paternal contributions that involve more than just providing the haploid male genome. Therefore, the overall objective of this project was to determine the impact of morphologically abnormal spermatozoa on fertilization, subsequent embryonic development, and embryo quality at the cellular level. Four experiments used morphologically abnormal semen samples collected and cryopreserved from four Holstein bulls before (Pre) and after a scrotal insulation (PI) period of 48 h. Zygotes were cultured for 8 d when a developmental score was assigned to each embryo; subpopulations were subjected to either the TUNEL or caspase assays to determine apoptosis. In the final experiment pronuclear decondensation for presumptive zygotes was evaluated by differential interference contrast microscopy at 3 h time intervals from 6 to 18 h post in vitro insemination (hpi). Morphological evaluation of semen samples revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the PI samples in comparison with the Pre samples for Bulls I and Bull III (74 to 22.3% and 67.7 to 0.5 %, respectively) and the scrotal insulation effects persisted from the time of cleavage through blastocyst formation for Bulls I and III and corresponded with a similar decrease in blastocyst development for PI samples in experiment 1 regardless of which semen separation method was used. Likewise, the overall pronuclear decondensation rate for the PI zygotes of Bull I and III showed no increase over time and remained predominantly at PN1 stage (1.5 ± 0.17; 1.8 ± 0.22, respectively). In contrast, the development for Bull II and Bull IV were unaffected. The embryo quality assessment revealed that the caspase intensity increased significantly for both Bull I (217 ± 147) and Bull III (229 ± 98) for the PI embryo groups compared to those of Bull II (98 ± 115) and Bull IV (90 ± 111). In conclusion, the tested separation methods used seemed inadequate in their ability to provide potentially competent sperm for IVF. The decrease in embryonic development appears to be multifaceted and related to the changes in head shape morphology and we suggest the failure in normal pronuclear formation is associated with an absence of normal decondensation of the penetrating spermatozoon. The inability to consistently measure apoptosis in early stage embryos complicates the assessment of differences in embryo quality. These observations support the hypothesis of uncompensable seminal traits in IVF with abnormal spermatozoa and provide compelling evidence that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus is manifested during the early stages of fertilization.