Pre-freezing selection of Holstein bull semen with the BoviPure colloid as double- or single-layer centrifugation improves the post-thawing quality.

Artificial insemination (AI) is critical for breeding in the dairy industry. High-merit bulls can present low freezability, hampering genetic dissemination. Thawed semen can be improved using density gradient centrifugation (DGC) with colloids, but little information deals with the pre-freezing application. Thus, the BoviPure colloid (optimized for bull spermatozoa) was tested for pre-freezing application as the usual double-layer (DLC) versus single-layer (SLC, quick and economical). Semen from twelve Holstein-Friesian bulls was extended with OPTIXcell extender, frozen (Control), or processed by SLC or DLC and frozen. Sperm were assessed pre-freezing for motility and viability and post-thawing (directly and after 4 h 38 °C) for apoptosis, capacitation status, acrosomal damage, mitochondrial activity, cytoplasmic and mitochondrial reactive oxygen species (ROS), and chromatin status (SCSA for DNA fragmentation and chromatin compaction and monobromobimane, mBBr, for disulfide bridges evaluation). The DGC improved parameters post-thawing (e.g., 57.5%±10.1 motility vs. control 53.3% ± 11.2) at the cost of sperm loss (sperm recovery of DGC 14.4% ± 2.5 and SLC 17.4% ± 2.5). DNA fragmentation (%DFI) decreased (0.21% ± 0.53 vs. control 1.30% ± 0.10), and SLC reduced chromatin compaction. A clustering procedure separated lesser (LF) and greater freezability (GF) bulls. LF samples were especially benefited by DGC, with SLC providing better post-thawing results for this group. In conclusion, pre-freezing DGC improved sperm parameters post-thawing, potentially improving the cryopreservation of low-freezability semen from high-merit bulls. SLC, quicker and economical, would be preferable since it showed similar or higher performance than DLC.