The goal of this study was to determine effects of trehalose (T) and Trolox (TX) additives on post-thaw viability of canine sperm after freezing. The sperm-rich portions of ejaculates obtained from 15 dogs were divided into three aliquots, which were diluted with Tris-based extender (TBE) (control), TBE + T (25 mM), and TBE + TX (1 mM) and put into straws. The samples were equilibrated at 4 °C for 1.5 hours, then frozen in liquid nitrogen vapour for 15 minutes and plunged into liquid nitrogen. Plasma membrane and acrosome integrity (PMAI) and high mitochondrial membrane potential (HMMP) were evaluated after thawing the straws in a water bath at 37 °C for 30 seconds. Motility, percentage of live spermatozoa, and hypo-osmotic swelling (HOS) were determined using phase-contrast microscopy. Motility (53.00 ± 6.46%), percentage of live spermatozoa (62.97% ± 6.84%), and HOS (40.40 ± 7.11%) of T were significantly higher (P <0.05) than the control (40.50 ± 4.76%, 50.63% ± 4.78%, and 30.47% ± 5.59%) and TX (40.50 ± 4.76%, 50.96 ± 5.84%, and 33.02% ± 8.77%) respectively. The PMAI and HMMP levels were higher ( P <0.05) in T (35.51±8.72 and 24.36±7.15%) than in the control (19.18 ± 3.49 and 12.04 ± 3.83%). The TX group (35.62 ± 8.21%) had significantly higher PMAI (P <0.05) than the control (12.04 ± 3.83%). Thus, addition of T protected canine spermatozoa against cryogenic injury. Nonetheless, additional dose-response trials are recommended to determine the effects of Trolox on the quality of canine semen more accurately.
"Experientia docet" - Experience is the best teacher