Lipid alterations by oxidative stress increase detached acrosomes after cryopreservation of semen in Holstein bulls

Theriogenology. 2023 Feb;197:37-45. doi: 10.1016/j.theriogenology.2022.11.036. Epub 2022 Nov 26.


The nearly exclusive use of cryopreserved semen in cattle breeding enables long shipping distances, higher storage times, quarantine to avoid germ transmission and easy dispersal of high genetic value bulls. Spermatozoa from bulls are well freezable and improvement of cryopreservation protocols over decades has led to high semen quality. However, there is still some loss of spermatozoa in each semen dose due to detached acrosomes after thawing. There are even individual bulls with extremely high numbers of detached acrosomes after cryopreservation, called "bad freezers". This study screened 1092 ejaculates from 59 Holstein bulls for the difference in detached acrosomes before and after cryopreservation (ΔAC). The individual bull influenced ΔAC (P < 0.001) and allowed selection for individuals with repeatedly low ΔAC (good freezers) or high ΔAC (bad freezers). Good freezers were superior to bad freezers in a thermo-resistance test (78.2% vs. 33.6% total motility, respectively, P = 0.047) and had higher non-return rates (NRR: 46.8% vs. 40.8%, respectively, P = 0.016). Since oxidative stress is one possible explanation for premature acrosome reaction, the radical reduction capacity of the seminal fluid was measured, finding that this parameter was reduced in bad freezer bulls during cryopreservation (P = 0.043). Analysis of lipid species in sperm cells by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a reduction of ether lipids and plasmalogens as well as an increase in formyl-lysophosphatidylcholines only within the bad freezers during cryopreservation (P = 0.043). In conclusion these findings show, that lipid alteration caused by oxidative stress is one essential reason for highly augmented acrosome reacted spermatozoa in bad freezer bulls. Therefore, increased use of antioxidants in the extender could be a possible starting point for developing individualized extenders for bad freezer bulls of high genetic value, in order to raise sperm quality after cryopreservation even in those bulls.

Keywords: Bull semen cryopreservation; Bull spermatozoa; Detached acrosomes; Membrane lipids; Oxidative stress.

MeSH terms

  • Acrosome
  • Animals
  • Cattle
  • Cryopreservation / methods
  • Cryopreservation / veterinary
  • Lipids
  • Male
  • Oxidative Stress
  • Semen Analysis / veterinary
  • Semen Preservation* / methods
  • Semen Preservation* / veterinary
  • Semen* / chemistry
  • Sperm Motility
  • Spermatozoa


  • Lipids