Desiccation tolerance in bovine sperm: A study of the effect of intracellular sugars and the supplemental roles of an antioxidant and a chelator q Ranjan Sitaula a,c , Heidi Elmoazzen c , Mehmet Toner c , Sankha Bhowmick a,b,c, * a Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, N. Dartmouth, MA, USA b Department of Mechanical Engineering, University of Massachusetts Dartmouth, N. Dartmouth, MA, USA c Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, MA, USA article info Article history: Received 11 August 2008 Accepted 17 March 2009 Available online 24 March 2009 Keywords: Desiccation Bovine Sperm Trehalose Sucrose Antioxidant Chelator Desferal Motility Membrane abstract Desiccation preservation holds promise as a simplified alternative to cryopreservation for the long term storage of cells. We report a study on the protective effects of intracellular and extracellular sugars during bovine sperm desiccation and the supplemental effects of the addition of an antioxidant (catalase) or a chelator (desferal). The goal of the study was to preserve mammalian sperm in a partially or completely desiccated state. Sperm loaded intracellularly with two different types of sugars, trehalose or sucrose, were dried with and without catalase and desferal and evaluated for motility and membrane integrity immediately after rehydration. Intracellular sugars were loaded using ATP induced poration. Drying was performed in desiccator boxes maintained at 11% relative humidity (RH). Results indicated that sperm exhibited improved desiccation tolerance if they were loaded with either intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1 mM desferal to the desiccation buffer. Though sperm motility after drying to low dry basis water fractions (DBWF) did not show significant improvement under any of the tested conditions, there was an increase in the sperm membrane integrity that could be retained after partial desiccation through the use of intracellular sugars and desferal. Ó 2009 Elsevier Inc. All rights reserved. Introduction Preservation of sperm for extended time periods has wide- spread applications in the fields of medicine, scientific research and in the dairy industry [24,25,44]. Cryopreservation, which has been the standard technique for the long-term preservation of spermatozoa, poses logistical and handling difficulties due to the necessity of storage in liquid nitrogen [24,37]. Desiccation preser- vation offers an attractive alternative to the cryopreservation tech- nique. The method, which mimics a naturally occurring phenomenon called anhydrobiosis, uses disaccharides, such as tre- halose and sucrose, to protect biologics and cells in a dehydrated state [7–10]. The potential for molecular immobilization without disruption or conformational changes to the structure of the bio- logical system is the key rationale for using sugars in preservation of biological systems [29]. As water is removed, sugars form a vis- cous matrix that reduces molecular mobility [10,19]. Anhydrobiot- ic engineering of mammalian cells requires that sugars be present on both sides of the plasma membrane in order to afford significant desiccation tolerance [16,1,6,45,11,15]. Therefore, an appropriate quantity of sugar must be loaded through the sperm plasma mem- brane. Various techniques have been used to load sugars into the cells [16,1,6,45,11]. These methods include thermal poration [23], electroporation [42], ATP poration [15] and the use of a-hemolysin to create transient non selective pores in cell membranes [16,1]. Though desiccation preservation offers an attractive alterna- tive to cryopreservation, there has been limited success in achieving dry storage of sperm using intracellular sugars [24,37]. Genetic integrity has been successfully preserved, but motility has not [4,34,30]. Although reconstituted freeze-dried spermatozoa can be used to fertilize mammalian oocytes by using intracytoplasmic sperm injection (ICSI) [25,26,46], little success has been achieved in preserving motility and membrane integrity after recovery from dehydrated states. After the suc- cessful recovery of fowl sperm post desiccation by Polge et al. [39], there has been a limited success in recovering motile sperm [40,36,47,28]. Previous studies have added glycerol to egg yolk– citrate buffer and then performed various freeze-drying proto- cols. Post rehydration motility has consistently been lost for sperm dried to moisture content fell below 10% with no storage survival. Studies without cytotoxic glycerol in the freeze-drying media have also shown similar trends [18]. 0011-2240/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2009.03.002 q This work was funded by a grant from ABS Global, Deforest, WI. * Corresponding author. Address: 285 Old Westport Road, Room # Textile 210, N. Dartmouth, MA 02747, USA. E-mail address: sbhowmick@umassd.edu (S. Bhowmick). Cryobiology 58 (2009) 322–330 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo