Differential protein expression in chicken spermatozoa before and after freezing–thawing treatment

• Freezing–thawing decreases the motility and viability of chicken sperm.
• The change in chicken sperm proteins after freezing–thawing was analyzed.
• The levels of 55 protein spots differed after freezing–thawing treatment.

The biological characteristics of rooster sperm that has undergone freezing treatment remain elusive. This study analyzed the change in sperm proteins after freezing–thawing treatment by using a proteomic approach. Semen from three 36-wk-old L2 strain Taiwan country chickens were used. A qualifying ejaculate containing more than 80% motility and volume 200 μL was used for cryopreservation. The proteomic analysis explored 55 protein spots that differed significantly before and after freezing–thawing treatment (P < 0.05). Among the 55 protein spots, expression levels of 19 proteins decreased after treatment. Forty-five differentially expressed protein spots were identified and belong to 33 proteins. Results of gene ontology analysis revealed that most differentially expressed proteins were involved in molecular function of the cellular metabolism process (28%) and cellular carbohydrate metabolism process (15%), and were associated with molecular function of oxidoreductase activity (19%) and protein binding (18%). The differentially expressed proteins before and after freezing–thawing treatment, including fructose-bisphosphate aldolase C, triosephosphate isomerase, aconitate hydratase, tubulin and outer dense-fiber protein, are associated with sperm energy metabolism and flagellum structure. In conclusion, freezing–thawing treatment significantly affects the expression of proteins related to sperm metabolism and structure in chicken spermatozoa. The differing levels of these proteins could be valuable for further enhancing the fertility of frozen–thawed chicken spermatozoa.