Low-density lipoprotein supplementation improves the quality of Holstein bulls' insemination doses

Despite the routine use of cryopreservation in dairy cattle, the subsequent deterioration in the quality of bull insemination doses (IDs) is an incentive to refine cryopreservation protocols, including the composition of the ID. The aim of this study was to examine, using a flow cytometry assay, whether the selected concentrations of low density lipoproteins (LDLs) extracted from hen-egg yolk may improve the post-thaw quality of the ID diluted with a plant-based extender. In total, 30 ejaculates from five sires (six trials per animal) were collected and processed at the Artificial Insemination Centre. The effect of the low density lipoprotein supplementation to the AndroMed (R) diluent at 4 and 8% (v/v) on the cryopreservation efficiency was tested. The effects of the modified extenders were determined on different sperm subpopulations by simultaneous quadruple staining for flow cytometry as the per-centage of sperm with intact plasma membrane and acrosome (PMAI), PMAI sperm showing high mitochondrial membrane potential (HMMP), sperm with plasma membrane damage (PMD), and sperm with acrosome damage (AD). It was observed that the 8% LDL treatment was more effective (P < 0.05) in preserving all the analysed param-eters than AndroMed (R) without any supplementation. The supplementation of the AndroMed (R) extender with 8% LDL resulted in the most optimal values of the PMAI (30.61 +/- 1.13), HMMP (68.81 +/- 1.25), PMD (68.69 +/- 1.14), and AD (38.36 +/- 1.13) compared to the control treatment (24.86 +/- 1.13 for PMAI, 47.79 +/- 1.25 for HMMP, 74.77 +/- 1.14 for PMD, and 42.83 +/- 1.13 for AD. The results of the study also demonstrated a synergistic positive effect of the LDL and soybean lecithin-based diluent on the spermatozoa post-thaw quality and resilience, based on 2 h long incubation. In conclusion, the soybean based semen extender treatment with LDL represents a beneficial tool to mitigate the detrimental effect of cryopreservation.