Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant Nikorn Thongtip a,b,* , Jumnian Saikhun c , Mangkorn Damyang a , Sittidet Mahasawangkul d , Piyawan Suthunmapinata a , Manoch Yindee a , Apisek Kongsila a , Tawepoke Angkawanish d , Sarun Jansittiwate d , Waroot Wongkalasin e , Worawidh Wajjwalkul a , Yindee Kitiyanant c , Kanok Pavasuthipaisit c , Anuchai Pinyopummin a a Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom 73140, Thailand b Center for Agricultural Biotechnology, Kasetsart University, Nakhon Pathom 73140, Thailand c Department of Anatomy, Faculty of Science, Institute of Science and Technology for Research and Development, Mahidol University, Nakhon Pathom 73170, Thailand d Thai Elephant Conservation Center, Forest Industry Organization, Lampang, Thailand e Asian Elephant Foundation, Phayathai, Bangkok 10400, Thailand Received 8 May 2003; received in revised form 18 August 2003; accepted 29 November 2003 Abstract Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility  60%), and were used for cryopreservation studies. Semen were frozen in TEST þ glycerol, TEST þ DMSO, HEPT þ glycerol, or HEPT þ DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42:0  4:3%; EM2: 26:0  17:3%) and the percentage of membrane and acrosome intact spermatozoa Theriogenology 62 (2004) 748–760 * Corresponding author. Tel.: þ66-34-579-0058; fax: þ66-34-351405. E-mail address: fvetnit@ku.ac.th (N. Thongtip). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.11.021