Addition of butylated hydroxytoluene (BHT) in tris-based extender improves post-thaw quality and motion dynamics of dog spermatozoa

Abstract

The aim of this study was to evaluate the interaction of different concentrations of butylated hydroxytoluene (BHT) in a tris-based extender on semen quality parameters in post-thawed dog semen. Twenty-four ejaculates were collected from eight male Beagle dogs using an artificial vagina. Pooled semen was diluted with a tris-based extender supplemented with 0 (control), 0.5, 1.0, 1.5, and 2.0 mM BHT, at a final concentration of 200 × 10⁶ spermatozoa/mL. After thawing, sperm samples were assessed for motility parameters (CASA), membrane integrity (SYBR-14/PI), acrosome integrity (FITC-PNA), mitochondrial activity (JC-1/PI), malondialdehyde (MDA) concentration, and glutathione peroxidase (GPx) activity. The total motility, progressive motility, and average path velocity of the frozen-thawed sperm were significantly higher in the BHT1.5 group than in the control and the other sample groups (P < 0.05). Higher values of straight-line velocity, curvilinear velocity, amplitude of the lateral head displacement, and linearity were observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (P < 0.05). The BHT1.0 and BHT1.5 groups had higher percentages of straightness and acrosome integrity than the other groups (P < 0.05). Beat cross frequency, plasma membrane integrity, and GPx activity of the BHT1.5 and BHT2.0 groups were higher than those of the control (P < 0.05). A lower concentration of MDA was observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (BHT0) (P < 0.05). Our results indicate that 1.5 mM BHT is the optimal concentration for improving the post-thaw quality of canine spermatozoa.

Keywords: Antioxidants; Butylated hydroxytoluene (BHT); Cryopreservation; Dog sperm; Sperm quality.