Effects of Equex, one- or two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa

The objectives of the present study were to evaluate the effects of adding Equex to a TRIS-extender, diluting the semen in 1 or 2 steps, freezing acco rding to 2 methods, thawing at 2 rates, and the interactions between these treatments, on the post-thaw survival of dog spermatozoa at 38 degreesC. Te n ejaculates were obtained from 8 dogs. Each ejaculate was centrifuged, and the seminal plasma was discarded. Each sperm pellet was diluted with 2 mt of a TRIS-glucose-egg yolk extender containing 3% glycerol (Extender 1 [Ext -ll). Ejaculates were then pooled (9 x 10(9) spermatozoa), and Ext-l was ad ded to obtain 200 x 10(6) spermatozoa/ml. The semen pool was carefully mixe d and divided into aliquots, and processed according to a 2 x 2 x 2 x 2 fac torial design to evaluate the effects of 1) adding the same volume of a sec ond TRIS-glucose-egg yolk extender with 7% glycerol that contained (Ext-2-E ) or didn't contain (Ext-2) 1% of Equex STM Paste (final concentration of s permatozoa 100 x 10(6) spermatozoa/ml, glycerol 5%, Equex 0% [Ext-2] or 0.5 % [Ext-2-E]); 2) diluting the semen in 1 step (adding Ext-2 or Ext-2-E befo re equilibration) or in 2 steps (adding Ext-2 or Ext-2-E after equilibratio n, just before the freezing operation!; 3) freezing the straws horizontally in a styrofoam box 4 cm above liquid nitrogen (LN2) or by lowering them ve rtically into a LN2 tank in 3 steps; and 4) thawing at 70 degreesC for 8 se c or at 37 degreesC for 15 sec. A total of 16 treatment combinations were e valuated. Sperm motility was evaluated after thawing and at l-h intervals d uring 7 h of incubation at 38 degreesC by subjective examination and by usi ng a CASA-system. Plasma membrane integrity and acrosomal status were evalu ated simultaneously at I, 3 and 6 h post-thaw using a triple fluorescent st aining procedure and flow cytometry. The best post-thaw survival and thermo resistance of spermatozoa was obtained when Equex was present in the extend er (P<0.0001); the semen dilution was performed in 2 steps instead of 1 (P< 0.0001); the freezing was carried out using the box instead of the tank (P< 0.05); and the straws were thawed at 70<degrees>C for 8 sec instead of at 3 7 degreesC for 15 sec (P<0.0001). (C) 2000 by Elsevier Science Inc.