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.

Extension of the equilibration period up to 24 h maintains the post-thawing quality of Holstein bull semen frozen with OPTIXcell®.

Semen cryopreservation in bovine livestock is well established, but logistics often require deviations from standard protocols. Extending the equilibration time to the following day is convenient in many situations. To improve our knowledge of the effects of this modification, we studied the post-thawing and post-incubation (4 h, 38 °C) sperm quality after freezing with 4 or 24-h extension in the OPTIXcell extender by using an ample panel of analyses: CASA for motility; flow cytometry for viability, physiology, oxidative stress, and chromatin parameters (DNA fragmentation, chromatin compaction, and thiol groups status); and spectrometry for malondialdehyde production. Semen was obtained from 12 Holstein bulls. The 24-h equilibration time showed few significant effects, with only a tiny decrease in progressive motility and a positive impact on chromatin structure. The incubation removed some of these effects, with the pattern for chromatin compaction remaining the same. No detrimental oxidative stress or increase in apoptotic or capacitation markers was detected. Additionally, the individual bull interacted with the effects of the incubation and the equilibration, especially regarding the chromatin status. Whereas this interaction did not critically affect sperm quality, it could be relevant in practice. Bull fertility as non-return rates (NRR56) was associated with some sperm parameters (especially with an improved chromatin structure) but not in the 4-h post-thawing analysis. Our study supports that extending the equilibration time by at least 24-h is feasible for bull semen freezing with the OPTIXcell extender.