Glycine and betaine improve the quality of cryopreserved sika deer sperm

Sperm cryopreservation imposes osmotic stress due to the addition and removal of high-concentration cryoprotectants. Whether organic osmolytes can alleviate this stress to improve cryopreservation efficiency remains unclear. Here, we evaluated the effects of glycine and betaine, supplemented as organic osmolytes throughout the cryopreservation process, on the quality and developmental potential of cryopreserved sika deer sperm. Pooled semen samples from five healthy adult stags were randomly allocated to four groups: a control (base extender) and three treatment groups supplemented with glycine and betaine at 6 mM + 200 μM, 10 mM + 500 μM, and 14 mM + 800 μM, respectively. The addition of 10 mM glycine and 500 μM betaine significantly improved post-thaw sperm quality, including enhanced total motility (TM), progressive motility (PM), average path velocity (VAP), curvilinear velocity (VCL), plasma membrane and acrosome integrity, and fertilization capacity, while reducing reactive oxygen species (ROS) levels compared with the control and other treatments. These findings suggest that glycine and betaine at 10 mM and 500 μM, respectively, may mitigate cryo-injury and enhance cryosurvival in sika deer sperm, offering a promising strategy for improving semen cryopreservation and artificial insemination protocols. • Glycine and betaine may alleviate osmotic and oxidative damage during sperm cryopreservation. • Both glycine and betaine significantly improved post-thaw sperm quality and fertilization capacity. • Organic osmolytes help sperm maintain volume homeostasis under cryopreservation-induced osmotic stress.