title: Cholesterol-loaded cyclodextrin improves ram sperm cryoresistance in skim milk-extender.
abstract: Cholesterol-loaded cyclodextrin (CLC) is known to improve ram sperm cryosurvival. This study expands on previous research to: (1) determine the mechanism by which CLC improves ram sperm cryosurvival and (2) compare the efficiency of a novel, skim milk-based extender containing CLC to a traditional egg yolk-based extender. Hypothesis #1 was that CLC enhances membrane cholesterol content to increase the resistance of ram sperm to cold and osmotic stress, thereby improving cryosurvival. We first assessed the ability of fresh sperm treated with CLC to withstand cold shock. Second, fresh sperm were treated with CLC to evaluate their tolerance to osmotic stress. Third, to confirm that cholesterol is incorporated into the sperm using CLC, we quantified sperm cholesterol. To test Hypothesis #2 that CLC is most effective in a medium without competing cholesterol, we compared sperm cryosurvival and fertility in skim milk-based extender containing CLC versus in a traditional egg yolk-based freezing extender without CLC. Our data confirmed that CLC treatment improves ram sperm cold shock and osmotic stress resistance, and augments sperm cholesterol content. Semen in skim milk-based extender containing CLC prior to freezing, had more motile sperm with intact acrosomes after thawing compared to semen in egg yolk-based extender. In contrast, sperm plasma membrane integrity and in vivo fertility of the semen cryopreserved in the skim milk-based extender with CLC did not differ from semen that was cryopreserved in egg yolk-based extender. Further research is warranted to combine CLC with other cryoprotection strategies or to modify the insemination protocol.
authors.affiliation: Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle, Département des Sciences Animales, Université Laval, Québec, QC G1V 0A6, Canada.
authors.forename: Vianney M
authors.initials: VM
authors.lastname: Salmon
authors.affiliation: Département des Sciences Animales, Université Laval, Québec, QC G1V 0A6, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Castonguay
authors.affiliation: Département des Sciences Animales, Université Laval, Québec, QC G1V 0A6, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Demers-Caron
authors.affiliation: Département d'obstétrique, Gynécologie et Reproduction, Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle, Santé de la mère et de l'enfant, Centre de Recherche du CHU de Québec, QC G1V 4G2, Canada.
authors.forename: Pierre
authors.initials: P
authors.lastname: Leclerc
authors.affiliation: Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle, Département des Sciences Animales, Université Laval, Québec, QC G1V 0A6, Canada. Electronic address: janice.bailey@fsaa.ulaval.ca.
authors.forename: Janice L
authors.initials: JL
authors.lastname: Bailey
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000494:pharmacology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D003451:Cryoprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D003505:Cyclodextrins / Q000494:pharmacology*; D004530:Egg Yolk / Q000737:chemistry / Q000502:physiology; D005260:Female; D005298:Fertility / Q000187:drug effects; D005615:Freezing; D008297:Male; D008892:Milk / Q000737:chemistry / Q000502:physiology; D011247:Pregnancy; D012661:Semen / Q000187:drug effects; D055101:Semen Analysis / Q000662:veterinary; D012662:Semen Preservation / Q000379:methods* / Q000662:veterinary; D012756:Sheep
chemical_list: D003451:Cryoprotective Agents; D003505:Cyclodextrins; D002784:Cholesterol
keywords: Artificial insemination; Cholesterol; Cold shock; Cyclodextrin; Osmotic tolerance