title: Semen cryopreservation for the creation of a Spanish poultry breeds cryobank: optimization of freezing rate and equilibration time.
abstract: A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following: 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the medium-rate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds.
authors.affiliation: Departamento Reproducción Animal, INIA, 28040 Madrid, Spain. moreno@inia.es
authors.forename: J
authors.initials: J
authors.lastname: Santiago-Moreno
authors.forename: C
authors.initials: C
authors.lastname: Castaño
authors.forename: A
authors.initials: A
authors.lastname: Toledano-Díaz
authors.forename: M A
authors.initials: MA
authors.lastname: Coloma
authors.forename: A
authors.initials: A
authors.lastname: López-Sebastián
authors.forename: M T
authors.initials: MT
authors.lastname: Prieto
authors.forename: J L
authors.initials: JL
authors.lastname: Campo
mesh_terms: D000818:Animals; D002645:Chickens / Q000235:genetics / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D005260:Female; D005306:Fertilization; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013030:Spain; D013094:Spermatozoa / Q000502:physiology*; D013997:Time Factors