The dynamics of sperm DNA stability in Asian elephant (Elephas maximus) spermatozoa before and after cryopreservation P. Imrat a , M. Hernandez b , S. Rittem b,c,d , N. Thongtip c,d,e , S. Mahasawangkul f , J. Gosálvez g , W.V. Holt b, * a Faculty of Veterinary Medicine, Utrecht University, Yalelaan 114, 3,584 CM Utrecht, the Netherlands b Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK c Center for Agricultural Biotechnology, Kasetsart University, Nakhonpathom 73140, Thailand d Center of Excellence on Agricultural Biotechnology, (AG-BIO/PERDO-CHE), Bangkok, Thailand e Department of Large Animal and Wildlife Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Nakhonpathom 73140, Thailand f the National Elephant Institute, the Forest, Industry Organization, Lampang 52190, Thailand g Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 8049, Spain Received 12 April 2011; received in revised form 5 October 2011; accepted 8 October 2011 Abstract The purpose of this study was to investigate the occurrence of sperm DNA fragmentation in Asian elephant (Elephas maximus) spermatozoa at various processing stages before and after cryopreservation. Five semen samples from four elephants were assessed at four different stages during processing; after (1) collection and reextension in TEST-egg yolk; (2) cooling to 5 °C; (3) equilibration for 1 h with glycerol; (4) thawing. An experimental approach was adopted that allowed comparisons of DNA fragmentation rates developed after the various processing stages. For this, spermatozoa were incubated in TEST-yolk media at 37 °C for 0, 4, 8, 24 and 48 h, and sperm DNA fragmentation rates were estimated using an elephant-specific Halosperm procedure. Incubation at 37 °C induced a rapid increase in DNA fragmentation, and significant differences between males were observed. The overall rate of increase over 4 h was estimated at about 5% per hour, and no significant changes to this rate were observed at the different processing stages, even, including the post-thaw samples. As semen quality of the five ejaculates was relatively poor, the basic semen parameter data were compared with nine different samples collected 11 mo earlier to see whether the tested samples were atypical or representative of the population, As there was no significant difference between the two sets of samples, it is believed that the samples tested for DNA stability were not unusually sensitive. These results suggest that Asian elephant spermatozoa are more susceptible to DNA fragmentation than spermatozoa of other mammals. © 2012 Elsevier Inc. All rights reserved. 1. Introduction The Asian elephant (Elephas maximus) is on Appen- dix I of the Convention on International Trade in En- dangered Species and is listed as endangered in the IUCN Red List of Threatened Species. Throughout the world, elephant populations are facing extinction, mainly because of human encroachment on their natu- ral habitats and geographic isolation of small popula- tions. In Thailand, despite the Asian elephant having been a national symbol since ancient times, the number of both wild and captive elephants is also in steep decline [1]. Although there are still a significant num- * Corresponding author. Tel: +44 207 6630; fax: +44 586 2870. E-mail address: bill.holt@ioz.ac.uk (B. Holt). Available online at www.sciencedirect.com Theriogenology 77 (2012) 998 –1007 www.theriojournal.com 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.10.007