This experiment was conducted to determine the most suitable glycerol concentration (3 or 6%) and/or non-penetrating cryoprotectants (trehalose and sucrose) for the cryopreservation of buffalo semen, with the aim of enhancing the cryopreservation protocol. Semen of Egyptian buffalo were pooled and diluted with eight Tris extenders supplemented with either 6% glycerol (control group, GL6), 3% (low level, GL3), sucrose (SU, 50 mM), trehalose (TR, 50 mM), 6% glycerol together with 50 mM of sucrose (GL6SU) or 50 mM of trehalose (GL6TR), and 3% of glycerol together with 50 mM of sucrose (GL3SU) or 50 mM of trehalose (GL3TR), then frozen following the standard protocol. Findings indicated that GL3 extender resulted in the highest values of progressive motility, sperm kinematics, sperm membrane integrity, and viability of post-thawed semen (37 °C for 30 s). On the contrary, the Tris extender enriched only with SU and TR groups had the lowest values of sperm quality compared to the other groups (p < 0.05). All GL supplemented groups showed higher intact acrosome levels and lower detached acrosome and dead sperm with intact acrosome compared to those with TR and SU alone (p < 0.05). A significant increase in viable sperm was observed in the GL3, GL6, and GL3SU groups compared to the other groups (p < 0.05). The Tris extender supplemented with low glycerol (3%) significantly reduced the levels of MDA. In the in vivo fertility trial, it was shown that the pregnancy rate was higher in the GL6SU group (72%) than in the GL3SU group (68%; p > 0.05). Collectively, these results suggest that there is potential in using low glycerol (3%) as a cryoprotective agent in the medium for buffalo sperm cryopreservation without significant adverse effects compared to the addition of 6% glycerol. This study supported the sustainability of materials used in assisted reproductive technology by reducing the glycerol content in the freezing medium. Further research is needed to confirm this hypothesis.
