title: 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 x 10(6), 100 x 10(6), 200 x 10(6) and 400 x 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 degrees C. Fifteen ejaculates were collected from 12 dogs and pooled. The semen pool was divided into 4 aliquots containing respectively 4,200 x 10(6), 2,100 x 10(6), 1,050 x 10(6) and 525 x 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 degrees C for 8 sec and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38 degrees 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 degrees 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 x 10(6) spermatozoa/mu. The integrity of sperm plasma membranes after 18 h incubation was higher (P<0.05) in samples frozen with 200 x 10(6) and 400 x 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 x 10(6) spermatozoa/mL was diluted immediately after thawing at 1:4 dilution rate.
authors: Department of Obstetrics and Gynecology, Center for Reproductive Biology, Swedish University of Agricultural Sciences, Uppsala.
authors: A
authors: A
authors: Peña
authors: C B
authors: CB
authors: Linde-Forsberg
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D001578:Benzopyrans; D002462:Cell Membrane / Q000502:physiology; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004285:Dogs / Q000502:physiology*; D004542:Ejaculation; D005434:Flow Cytometry / Q000662:veterinary; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D037102:Lectins / Q000737:chemistry; D016014:Linear Models; D008297:Male; D008858:Microscopy, Phase-Contrast / Q000662:veterinary; D009284:Naphthols / Q000737:chemistry; D011419:Propidium / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D001578:Benzopyrans; D005456:Fluorescent Dyes; D037102:Lectins; D009284:Naphthols; D012235:Rhodamines; C068301:carboxy-seminaphthorhodaminefluoride; D011419:Propidium; D016650:Fluorescein-5-isothiocyanate