title: Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size.
abstract: BACKGROUND
  An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions).
  
  
  OBJECTIVE
  To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons.
  
  
  METHODS
  Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion of spermatozoa with undamaged chromatin) was assessed before and after SLC.
  
  
  RESULTS
  All parameters of sperm quality examined were significantly better in sperm samples after SLC than in their unselected counterparts (P<0.001 for each parameter). The yield of spermatozoa obtained after SLC was influenced by the type of extender used and also by the concentration of spermatozoa in the original ejaculate, with fewer spermatozoa being recovered when the loading dose contained a high concentration of spermatozoa. The optimal concentration was approximately 100 × 10⁶/ml. Sperm concentration in the samples loaded on to the colloid influenced the sperm yield while the type of semen extender affected sperm quality and survival. Furthermore, the scaled-up SLC method was found to be suitable for use with a range of ejaculates, with similar sperm kinematics being observed for standard and scaled-up preparations.
  
  
  CONCLUSIONS
  SLC consistently improved the quality of stallion sperm samples from a large number of ejaculates. The method could be scaled-up, allowing larger volumes of ejaculate to be processed easily from a wide range of stallions.
authors.affiliation: Department of Reproduction, Division of Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden. jane.morrell@kv.slu.se
authors.forename: J M
authors.initials: JM
authors.lastname: Morrell
authors.forename: H
authors.initials: H
authors.lastname: Rodriguez-Martinez
authors.forename: A
authors.initials: A
authors.lastname: Johannisson
mesh_terms: D000818:Animals; D002498:Centrifugation / Q000379:methods / Q000662:veterinary*; D006736:Horses / Q000502:physiology*; D008297:Male; D012189:Retrospective Studies; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013048:Specimen Handling / Q000379:methods / Q000662:veterinary*; D013076:Sperm Count / Q000662:veterinary; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000502:physiology*