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The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA

Luna Gutiérrez-Cepeda1*, Álvaro Fernández1, Francisco Crespo2, Miguel Ángel Ramírez3, Jaime Gosálvez4 and Consuelo Serres1

Author Affiliations

1 Animal Medicine and Surgery Deparment, Veterinary Faculty, UCM, Avda. Puerta de Hierro s/n, Ciudad Universitaria, 28040, Madrid, Spain

2 Centro Militar de Cría Caballar, (FESCCR-Ministerio de Defensa), Ávila, Spain

3 Halotech DNA, S.L. UAM, Madrid, Spain

4 Biology Department, Genetic Unity, UAM, Madrid, Spain

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Acta Veterinaria Scandinavica 2012, 54:72  doi:10.1186/1751-0147-54-72

Published: 5 December 2012



Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics).


Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05).


These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics.

Colloidal centrifugation; Equine sperm; DNA fragmentation; Cryopreservation; Equipure®