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Effects of a dimethylformamide-glycerol combination on freezing behavior, post-thaw sperm quality, and sperm-oocyte
Manuel Soria1, Juan Ochoa-Guillén1, David Álvarez-Palacios1
1Laboratorio de Biotecnología de la Reproducción Animal, Facultad de Ciencias Agropecuarias, Universidad de Cuenca, EC010205, Cuenca, Ecuador.
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.
