Cryopreservation of turkey semen by the pellet method: Effects of variables such as the extender, cryoprotectant concentration, cooling time and warming temperature on sperm quality determined through principal components analysis Nicolaia Iaffaldano a, *, Luca Romagnoli b , Angelo Manchisi a , Maria Pina Rosato a a Department of Animal, Plant and Environmental Science, University of Molise, via De Sanctis, 86100 Campobasso, Italy b Department of Scienze Economiche Gestionali e Sociali, University of Molise, via De Sanctis, 86100 Campobasso, Italy Received 30 August 2010; received in revised form 30 March 2011; accepted 19 April 2011 Abstract This study was designed to identify the best pellet cryopreservation procedure for the cryosurvival of turkey semen among 192 different treatments established by variations and permutations of seven conditions used in the freezing/thawing process. These conditions were: diluent (IGGKPh, SPh or Tselutin); dilution rate (1:3 vs. 1:4); cooling time (45 vs. 60 min); dimethylacetamide (DMA) concentration as cryoprotectant (6 vs. 8%); equilibration time in DMA (1 vs. 5 min); semen drop volume (50 vs. 80 L) and thawing temperature (60 vs. 75 °C). Through principal components analysis (PCA), post-thaw sperm quality data (mobility, viability and membrane functional integrity) were reduced to a single output variable (Sperm Quality) indicating overall post-thaw semen quality. All treatments induced a significant reduction in semen quality after warming (P  0.01), though one set of seven conditions, or treatment, was identified by PCA to generate the highest Sperm Quality score and a further five treatments yielded a score not significantly different (P  0.05) from this best score. Although still not fulfilling the requirements for commercial application, our findings serve to identify the critical steps in turkey sperm cryopreservation that need to be assessed in future studies. © 2011 Elsevier Inc. All rights reserved. Keywords : Turkey Semen; Cryopreservation; PCA 1. Introduction Semen cryopreservation has long been used in ani- mal production and genetic improvement programs as well as for preserving genetic biodiversity. However, despite the good progress made in the cryopreservation of semen in cattle, advances in other livestock species, such as pigs or sheep [1] have been less dramatic. The poultry industry has not been able to take advantage of the opportunities offered by frozen semen, because of both the high costs of storing and preparing frozen ejaculates for use and the low fertility levels achievable with frozen/thawed spermatozoa [2,3]. Despite this rather pessimistic scenario, a range of protocols have been developed by researchers and their results have been reviewed by many authors [3-9]. These authors have described a high sensitivity of avian spermatozoa to cryopreservation-induced damage with the conse- quence of highly variable fertility rates obtained with frozen/thawed poultry semen that are insufficiently re- * Corresponding author. Tel.: +39 0874 404697; fax: +39 0874 404855. E-mail address: nicolaia@unimol.it (N. Iaffaldano). Available online at www.sciencedirect.com Theriogenology 76 (2011) 794 – 801 www.theriojournal.com 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.04.012