Effects of the Tris, Tes, or skim milk based extender on in vitro parameters of ram spermatozoa during liquid storage

• Ram semen was preserved at 4 °C in Tris, Tes, or milk based extender.
• The common parameters including motility, moving velocity, hypo-osmotic swelling capability, acrosome status, membrane integrity, distribution of phosphatidylserine, and mitochondrial membrane potential were assessed.
• The protection of Tris or Tes based extender is higher than milk.
• Tris can be substituted by Tes in liquid storage of semen.

Cryopreservation can seriously damage structure and physiological function of mammalian spermatozoa. However, liquid storage at reduced temperature may be an alternative of cryopreservation if artificial insemination is performed in relatively short time. In this study, effects of the Tris-hydroxymethyl aminomethane (Tris), N-Tris (hydroxymethyl) -methylaminoethane-sulfonic acid (Tes), or skim milk based extender on ram spermatozoa during liquid storage at 4 °C were assessed and compared. Semen samples from 6 adult Yunnan semi-fine wool rams with proven fertility were pooled, equally divided into 3 groups according to extenders, and preserved at 4 °C for various durations (0, 1, 3, 5, and 7 days). The motility, moving velocity, and hypo-osmotic swelling capability of spermatozoa were evaluated using a computer assisted sperm analyzer system. The acrosome status, membrane integrity, distribution of phosphatidylserine (PS), and mitochondrial membrane potential (MMP) were analyzed with flow cytometry. The results indicated that following liquid storage in the Tris or Tes based extender for 3 days, the motility, hypo-osmotic swelling capability, and MMP of spermatozoa did not show a significant decrease. Additionally, liquid storage in the Tris or Tes based extender for 24 h did not deleteriously affect the moving velocity, acrosome status, and membrane integrity of spermatozoa. However, after liquid storage for 24 h, the percentage of spermatozoa with membrane exposed PS was significantly increased. On the contrary, the negative effects of skim milk on the above parameters of ram spermatozoa became more serious compared to the Tris or Tes based extender after liquid storage for 24 h (P < 0.05). There was no significant difference existing between the Tris and Tes based extender as concerning the parameters evaluated in this study. In conclusion, the Tris or Tes based extender may be more suitable for liquid storage of ram spermatozoa at reduced temperature compared to the skim milk based extender. Tris can be substituted by Tes for liquid storage of ram semen. The future study should focus on assessment of molecular changes and fertility of ram spermatozoa after liquid storage.