Effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa

Abstract

The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 × 10 6, 100 × 10 6, 200 × 10 6 and 400 × 10 6 spermatozoa/mL) in 0.5-mL straws and diluting the thawed semen at 4 different rates (1:0, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38 °C. Fifteen ejaculates were collected from 12 dogs and pooled. The semen pool was divided into 4 aliquots containing respectively 4200 × 10 6, 2100 × 10 6, 1050 × 10 6 and 525 × 10 6 spermatozoa, which were centrifuged. Sperm pellets were rediluted with TRIS-glucose-egg yolk extender containing 5% glycerol and 0.5% of Equex STM Paste to obtain the designated sperm concentrations. The semen was frozen in 0.5-mL straws 4 cm above liquid nitrogen (LN2). The straws were thawed at 70 °C for 8 sec and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38 °C at rates of 1:0, 1:1, 1:2 and 1:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at 1-h intervals during 8 h of incubation at 38 °C. Plasma membrane integrity and acrosomal status were evaluated at 1, 3, 6, 12 and 18 h post-thaw using a triple-staining procedure and flow cytometry. For data pooled across the post-thaw dilution rate, motility was higher (P<0.001) in samples frozen with 200 × 10 6 spermatozoa/mL. The integrity of sperm plasma membranes after 18 h incubation was higher (P<.05) in samples frozen with 200 × 10 6 and 400 × 10 6 spermatozoa/mL. For data pooled across sperm concentration, samples diluted at a rate of 1:2 or 1:4 had better (P<0.001) motilities after 8 h of incubation than undiluted samples or those diluted at 1:1. The integrity of the sperm plasma membranes was higher (P<0.001) at increasing dilution rates. When the 16 treatments were compared, the best longevity was obtained when semen packaged at a concentration of 200 × 10 6 spermatozoa/mL was diluted immediately after thawing at 1:4 dilution rate.