Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen- thawed semen without affecting seminal kinetics T. Guimarães a, b , G. Lopes a, b , M. Pinto a , E. Silva a, c , C. Miranda a , M.J. Correia d , L. Damásio e , G. Thompson a, c , A. Rocha a, b, * a Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Portugal b Centro de Estudos de Ciência Animal (CECA/ICETA), University of Porto, Porto, Portugal c Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), University of Porto, Porto, Portugal d Fundação Alter Real, Alter do Chão, Portugal e Private Practitioner, Évora, Portugal article info Article history: Received 15 April 2014 Received in revised form 3 September 2014 Accepted 8 September 2014 Keywords: Stallion Semen Cryopreservation Bacteria Androcoll-E abstract Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen’s microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without pre- vious SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorganism species and total microbial load (TML) expressed in colony-forming units (CFU/mL) as well as frozen-thawed sperm kinetics were assessed in both groups. Additional analysis of the TML was performed, subdividing the frozen-thawed samples in “suitable” (total motility  30%) and “unsuitable” (total motility < 30%) semen for freezing programs, and comparing the C and SLC groups within these subpopulations. After thawing, SLC samples had less (P < 0.05) TML (88.65  10 2  83.8  10 2 CFU/mL) than C samples (155.69  10 2  48.85  10 2 CFU/mL), mainly due to a reduction of Enterococcus spp. and Bacillus spp. A relationship between post-thaw motility and SLC effect on ML was noted, as only in samples with more than 30% total motility was ML reduced (P < 0.05) by SLC (from 51.33  10 2  33.26  10 2 CFU/mL to 26.68  10 2  12.39  10 2 CFU/mL in “suitable” frozen-thawed semen vs. 240.90  10 2  498.20  10 2 to 139.30  10 2  290.30  10 2 CFU/mL in “unsuitable” frozen-thawed semen). The effect of SLC on kinetics of frozen-thawed sperm cells was negligible. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction It has been estimated that 20% of stallions produce semen that freezes well, 60% semen that freezes acceptably, and 20% of the stallions have semen that freezes poorly [1,2]. The mechanisms underlying the differences in cry- osensitivity between individuals are largely unknown [2], with those differences attributed to genetic factors [2,3], type of cryoprotectant utilized [3,4], and other nongenetic factors not yet elucidated [2]. Bacterial contamination is known to affect semen dur- ing cold storage [5]. Frozen-thawed semen contains bac- teria [6–8], and Hoogewijs et al. [9] hypothesized that a reduction in bacterial contamination would improve the * Corresponding author. Tel.: þ351 252 660 403; fax: þ351 252 661 780. E-mail address: arocha@mail.icav.up.pt (A. Rocha). Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.09.003 Theriogenology 83 (2015) 186–191