The effect of cryopreservation on the turkey (Meleagris gallopavo) semen proteome

Two-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry was employed to investigate the proteome alterations induced by equilibration and freezing/thawing processes, both in turkey spermatozoa and in extracellular fluid (ECF). The freezing/thawing process resulted in reduced semen quality parameters. Viability and motility decreased threefold (90.6 %-31.2 % and 76.0 %-26.7 %, respectively), while the proportion of live spermatozoa with intact mitochondrial membrane potential decreased fivefold (54.9 %-11.4 %). Additionally, oxidative stress increased sevenfold (10.5 %-68.8 %). A total of 146 differentially abundant protein spots were found between fresh and frozen/thawed spermatozoa, while 27 spots differentiated between fresh and frozen/thawed ECF. Immunofluorescence staining showed reduced signals of mitochondrial proteins, such as aconitate hydratase, alpha-enolase, glycerol-3-phosphate dehydrogenase, and triosephosphate isomerase, in the spermatozoa midpiece, as well as reduced signals of acrosin in the acrosome. Freezing/thawing affected mitochondrial energy metabolism, particularly the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. The maintenance of an acetyl-CoA pool to sustain TCA cycle activity in cryopreserved spermatozoa may be insufficient owing to disturbances in fatty acid beta-oxidation and/or aerobic glycolysis. Changes in the ECF primarily reflect the leakage of spermatozoa glycolytic enzymes. The freezing/thawing process alters motile cilium assembly, primarily affecting the spermatozoa axoneme and outer dense fibres. The initial step of fertilization may be disrupted by alterations in proteins involved in spermatozoa binding to the ovum. These findings extend our knowledge of the molecular and cellular mechanisms of cryodamage in turkey semen which is prerequisite for the improvement of semen preservation procedures.