Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca2+ concentration of dog spermatozoa

Abstract

The aims of the present study were to compare the effects of two commercial preparations (Equex STM Paste or Equex Pasta), whose active ingredient is sodium dodecyl sulphate (SDS), added to a Tris–egg yolk-based extender, on post-thaw sperm survival and longevity, as well as on the intracellular Ca2+ concentration of dog spermatozoa during incubation at 38°C. One ejaculate was collected from each of eight dogs. Each ejaculate was centrifuged, the semen plasma discarded, and the sperm pellet rediluted with a Tris–glucose–egg yolk extender containing 3% glycerol (Ext-1) at a sperm concentration of 200×106 spermatozoa (spz)/ml. The diluted semen was divided in three aliquots of equal volume and allowed to equilibrate for 1h at 4°C. After equilibration, the same volume of three different second extenders was added, respectively, to each of the three aliquots: (A) Ext-2A (same composition as Ext-1 except that it contained 7% glycerol and 1% Equex STM Paste), (B) Ext-2B (same composition as that of Ext-1 except that it contained 7% glycerol and 1% Equex Pasta), and (C) Ext-2 (Control: same composition as that of Ext-1 except that it contained 7% glycerol). Semen samples were packed in 0.5ml straws and frozen on a rack 4cm above liquid nitrogen (LN2) in a styrofoam box. Thawing was at 70°C for 8s. Sperm motility was evaluated after thawing and at 1h intervals for 5h at 38°C by subjective examination and by using a CASA system. Plasma membrane integrity and acrosomal status were evaluated at 1, 4 and 7h post-thaw using a triple staining procedure and flow cytometry. Intracellular Ca2+ concentration of live spermatozoa was evaluated by flow cytometry at 1, 4 and 7h post-thaw after co-loading the sperm cells with the Ca2+ indicators Fluo 3 AM and Fura Red AM, and with PI. Post-thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better (P<0.005) when Ext-2A (containing Equex STM Paste) was used. There was no difference between Ext-2B (containing Equex Pasta) and Ext-2 (Control). The mean intracellular Ca2+ concentration (arbitrary units) of cryopreserved spermatozoa (range: 0.23±0.12 to 1.26±0.46) was higher than that of fresh spermatozoa (0.13±0.06). When using Ext-2A, the live spermatozoa frequently (P=0.012) appeared divided in two subpopulations, with high (1.26±0.46) and low (0.27±0.09) intracellular Ca2+ content, respectively. When using Ext-2B or Ext-2, the live spermatozoa were more frequently seen in a single population with low intracellular Ca2+ concentration (0.30±0.35 and 0.23±0.12, for Ext-2B and Ext-2, respectively).