ABSTRACT
The present work aimed to assess the effect of equilibration time on post-thaw motility parameters of canine sperm frozen in three extenders: 6% low-density lipoproteins (LDL), 6% liposomes (LIPO), and 40% egg yolk plasma (EYP). A second experiment is aimed at evaluating the functional integrity of canine spermatozoa frozen in the three extenders at the best equilibration time found in the experiment one. In the first experiment, 20 ejaculates harvested from 7 dogs, were frozen in three extenders (LDL, LIPO, and EYP) after four equilibration times (30min, 1h, 3h, and 6h). The semen was evaluated after thawing using an image analyser (HT-IVOS 14.0). The 6h equilibration time gave better results of motility and progressive motility in the three studied extenders. (LDL: 58.9% vs. 42.7%; LIPO: 54.4% vs. 31.9%; EYP: 55.4% vs 40.5% for motility 6 vs. 1h). In the second experiment, 10 ejaculates taken from 6 dogs were frozen under the same conditions as the previous experiment, after 6h equilibration time. The integrity parameters of the spermatozoal membrane (hypo-osmotic swelling test, and SYBR14/propidium Iodide staining), acrosome (FITC-Pisium sativum Aglutinin staining), and DNA (acridine orange staining) were evaluated at three different stages: post-dilution (T0), post-equilibration, and post-thawing. Post-thaw results were as follows: membrane integrity (HOSt: 62;6% vs 58% vs 64.4%; SYBR14/IP: 63.6% vs 57.9% vs 64.8%); acrosome integrity (FITC-PSA: 79.4% vs 74% vs 76.2%) and DNA integrity (Acridine-orange: 98.9% vs 98.5% vs 98.7%) respectively for LDL vs. LIPO vs. EYP. No significant difference existed between the extenders tested; thus 6%LIPO and 40%EYP could be good candidates for replacement of 6%LDL in the protection of canine sperm during the freeze-thaw process without altering motility and integrity parameters.
Subject(s)
Cryopreservation/veterinary , Cryoprotective Agents/pharmacology , Dogs/physiology , Semen Preservation/veterinary , Sperm Motility/drug effects , Spermatozoa/drug effects , Acrosome/physiology , Animals , Cryopreservation/methods , Egg Yolk , Freezing , Lipoproteins, LDL , Male , Semen/drug effects , Semen Preservation/methods , Spermatozoa/physiologySubject(s)
Body Temperature , Circadian Rhythm , Adult , Female , Humans , Male , Surveys and Questionnaires , Time FactorsABSTRACT
Granulocytes were harvested from each of five healthy male volunteers once by continuous flow centrifugation with the IBM-Aminco Celltrifuge, and once by adhesion filtration leukapheresis with nylon fiber. Granulocyte recovery and purity were significantly better with the filtration leukapheresis system than with continuous flow centrifugation. Measurements of trypan blue dye exclusion and muramidase activity were similar to those in control granulocytes regardless of the method of isolation. Granulocyte-stimulated oxygen consumption was diminished in granulocytes prepared by the adhesion filtration method, but normal in those prepared by continuous flow centrifugation with the IBM-Aminco Celltrifuge.
