Effect of lactoferrin on ram sperm motility after cryopreservation

Objective: The objective of this study was to analyse the differentially abundant proteins caused by freeze-thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro. Methods: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 p.g/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma mem- brane integrity, mitochondrial activity and reactive oxygen species (ROS). Results: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 p.g/mL LTF compared to those in the control group. There was no significant difference in mito- chondrial activity between the 0 p.g/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 p.g/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05). Conclusion: The LTF is an important protein during cryopreservation, and the addition of 10 p.g/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.