title: Effect of equilibration time on the motility and functional integrity of canine spermatozoa frozen in three different extenders.
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.
authors.affiliation: Laboratory of Biotechnology and Reproductive Pathology, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France; Laboratory of Animal Reproduction Biotechnologies, SAAD DAHLAB University of Blida (U.Blida1), BP 270, Blida (09000) Algeria.
authors.forename: R
authors.initials: R
authors.lastname: Belala
authors.affiliation: Laboratory of Biotechnology and Reproductive Pathology, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France.
authors.forename: L
authors.initials: L
authors.lastname: Briand-Amirat
authors.affiliation: Laboratory of Biotechnology and Reproductive Pathology, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France.
authors.forename: L
authors.initials: L
authors.lastname: Vinciguerra
authors.affiliation: Laboratory of Biotechnology and Reproductive Pathology, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France.
authors.forename: D
authors.initials: D
authors.lastname: Tainturier
authors.affiliation: Laboratory of Animal Reproduction Biotechnologies, SAAD DAHLAB University of Blida (U.Blida1), BP 270, Blida (09000) Algeria.
authors.forename: R
authors.initials: R
authors.lastname: Kaidi
authors.affiliation: Department of Statistics, ONIRIS: The National Veterinary, Food agriculture, and food hygiene school of Loire Atlantique, BP 40706, 44307 Nantes, France.
authors.forename: C
authors.initials: C
authors.lastname: Thorin
authors.affiliation: Laboratory of Biotechnology and Reproductive Pathology, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France.
authors.forename: S
authors.initials: S
authors.lastname: Michaud
authors.affiliation: UR1268 Biopolymères Interactions Assemblages, Equipe Interfaces et Systèmes Dispersés, INRA, F-44316 Nantes Cedex 3, France.
authors.forename: M
authors.initials: M
authors.lastname: Anton
authors.affiliation: Laboratory of Biotechnology and Reproductive Pathology, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France. Electronic address: djemil.bencharif@oniris-nantes.fr.
authors.forename: D
authors.initials: D
authors.lastname: Bencharif
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004285:Dogs / Q000502:physiology*; D004530:Egg Yolk; D005615:Freezing; D008077:Lipoproteins, LDL; D008297:Male; D012661:Semen / Q000187:drug effects; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D003451:Cryoprotective Agents; D008077:Lipoproteins, LDL
keywords: Cryopreservation; Dog semen; Egg yolk plasma; Equilibration time; In-vitro fertility tests; Liposomes