Effects of a dimethylformamide–glycerol combination on freezing behavior, post-thaw sperm quality, and sperm–oocyte interaction in Arabian stallions

Background: Although glycerol (GLY) and dimethylformamide (DMF) are widely used cryoprotectant agents (CPAs) for freezing equine semen, their combined use remains less characterized.
Aims: To evaluate the effects of DMF, GLY, and their combination (DMF+GLY) on freezing behavior, sperm
kinematics, membrane integrity, and IVF-related functional endpoints in Arabian stallion sperm.
Methods: Twenty-four ejaculates from four stallions (six/male) were diluted in BotuSemen-Gold® extender supplemented with 5% DMF, 5% GLY, or 3% DMF + 3% GLY, packed into 0.5-ml straws, and frozen. Sperm–oocyte binding and pronuclear formation were assessed by heterologous IVF using in vitro–matured bovine oocytes and frozen–thawed sperm from each treatment.
Results: DMF+GLY exhibited intermediate seeding (at -9.0°C at 11 s) and apparent dissipation of latent heat fusion (6 s), suggesting a more balanced freezing behavior. DMF+GLY produced higher motility, curvilinear velocity, beat-cross frequency, and acrosomal integrity than GLY alone (P < 0.05), but these variables did not differ from DMF alone. However, sperm viability, straight-line velocity, and average path velocity were higher in DMF+GLY than both GLY and DMF alone (P < 0.05). DMF+GLY (n=240 oocytes) also increased sperm–oocyte binding at 2.5 h compared with DMF (n=258) or GLY (n=214) alone (P < 0.05), whereas pronuclear formation was not affected by CPA treatment (P > 0.05).
Conclusion: 3% DMF + 3% GLY improved freezing behavior, selected post-thaw sperm quality parameters, and sperm–oocyte binding under the conditions tested, although the higher total CPA concentration of this treatment should be considered when interpreting these findings.