Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics

Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen’s microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without previous SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorganism species and total microbial load (TML) expressed in colony-forming units (CFU/mL) as well as frozen-thawed sperm kinetics were assessed in both groups. Additional analysis of the TML was performed, subdividing the frozen-thawed samples in “suitable” (total motility ≥ 30%) and “unsuitable” (total motility < 30%) semen for freezing programs, and comparing the C and SLC groups within these subpopulations. After thawing, SLC samples had less (P < 0.05) TML (88.65 × 102 ± 83.8 × 102 CFU/mL) than C samples (155.69 × 102 ± 48.85 × 102 CFU/mL), mainly due to a reduction of Enterococcus spp. and Bacillus spp. A relationship between post-thaw motility and SLC effect on ML was noted, as only in samples with more than 30% total motility was ML reduced (P < 0.05) by SLC (from 51.33 × 102 ± 33.26 × 102 CFU/mL to 26.68 × 102 ± 12.39 × 102 CFU/mL in “suitable” frozen-thawed semen vs. 240.90 × 102 ± 498.20 × 102 to 139.30 × 102 ± 290.30 × 102 CFU/mL in “unsuitable” frozen-thawed semen). The effect of SLC on kinetics of frozen-thawed sperm cells was negligible.