title: Effects of glycerol and sugar mixing temperature on the morphologic and functional integrity of cryopreserved ram sperm.
abstract: Sperm deep freezing procedures for ram semen have considerable variations regarding the steps being employed for cooling, freezing, and addition of cryoprotectants. In this work, we evaluated the effects of the addition of glycerol and/or the disaccharides sucrose and trehalose to hypertonic diluents either before or after cooling from 30 °C to 5 °C in Merino Australian ram semen cryopreservation. Using optical and transmission electron microscopy techniques, we assessed that glycerol was beneficial to the cooling process independently of its addition at 30 °C or 5 °C in terms of sperm membrane integrity in different regions of the plasma membrane (acrosomal region, 14.5% higher integrity; postacrosomal region, 8.0% higher integrity [P < 0.01]; hypoosmotic swelling test [HOST], 10.8% higher integrity [P < 0.001]). Disaccharides were necessary for a better cryopreservation in liquid nitrogen, and the best procedure was their addition after cooling at 5 °C (12% higher sperm motility [P < 0.001]; 8% higher acrosome integrity, [P < 0.05]; 9.5% higher plasma membrane integrity assessed by HOST [P < 0.001]). Trehalose showed a greater preservation cryoprotectant capacity than sucrose, as indicated by sperm motility after thawing (8.1% greater [P < 0.01]) and by the integrity of the intermediate piece (20% greater [P < 0.05]). From these results, we conclude that the best procedure for ram semen cryopreservation in hypertonic disaccharide-containing diluents is the addition of glycerol and trehalose after the cooling process, at 5 °C.
authors.affiliation: Laboratorio de Teriogenología "Dr. Héctor H. Morello", Instituto de Biotecnología Agropecuaria del Comahue (IBAC). Facultad de Ciencias Agrarias, Universidad Nacional del Comahue, Cinco Saltos, Río Negro, Argentina.
authors.forename: V
authors.initials: V
authors.lastname: Pelufo
authors.affiliation: Laboratorio de Teriogenología "Dr. Héctor H. Morello", Instituto de Biotecnología Agropecuaria del Comahue (IBAC). Facultad de Ciencias Agrarias, Universidad Nacional del Comahue, Cinco Saltos, Río Negro, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Cinco Saltos, Río Negro, Argentina.
authors.forename: M F
authors.initials: MF
authors.lastname: López Armengol
authors.affiliation: Ministerio de Desarrollo Territorial, Gobierno de la Provincia del Neuquén, Neuquén, Argentina.
authors.forename: V
authors.initials: V
authors.lastname: Malcotti
authors.affiliation: Laboratorio de Teriogenología "Dr. Héctor H. Morello", Instituto de Biotecnología Agropecuaria del Comahue (IBAC). Facultad de Ciencias Agrarias, Universidad Nacional del Comahue, Cinco Saltos, Río Negro, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Cinco Saltos, Río Negro, Argentina.
authors.forename: A
authors.initials: A
authors.lastname: Venturino
authors.affiliation: Laboratorio de Teriogenología "Dr. Héctor H. Morello", Instituto de Biotecnología Agropecuaria del Comahue (IBAC). Facultad de Ciencias Agrarias, Universidad Nacional del Comahue, Cinco Saltos, Río Negro, Argentina. Electronic address: teriogenologia@hotmail.com.
authors.forename: E G
authors.initials: EG
authors.lastname: Aisen
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000737:chemistry / Q000494:pharmacology; D005615:Freezing; D005990:Glycerol / Q000737:chemistry / Q000494:pharmacology*; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D012756:Sheep / Q000502:physiology*; D013395:Sucrose / Q000737:chemistry / Q000494:pharmacology*; D014199:Trehalose / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D003451:Cryoprotective Agents; D013395:Sucrose; D014199:Trehalose; D005990:Glycerol
keywords: Cooling process; Frozen-thawed ram spermatozoa; Plasma membrane domains; Trehalose