Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender q Abozar Najafi a , Hossein Daghigh Kia a,⇑ , Hossein Mohammadi a , Mir Hossein Najafi b , Zaynab Zanganeh b , Mohsen Sharafi b,c , Felipe Martinez-Pastor d , Hamideh Adeldust e a Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran b Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran c Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran d INDEGSAL and Molecular Biology, University of León, León, Spain e Department of Animal Science, College of Agriculture, University of Kurdistan, Iran article info Article history: Received 7 January 2014 Accepted 10 May 2014 Available online xxxx Keywords: Ram semen Antioxidant Cysteamine Ergothioneine Cryopreservation abstract The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender. Ó 2014 Elsevier Inc. All rights reserved. Introduction The cryopreservation of spermatozoa has allowed the conserva- tion of genetic resources in sperm banks, the guarantee of a con- stant commercial supply of semen, and the development of breed improvement programs by means of the artificial insemination (AI) technique [9,25,30]. However, the use of AI in small ruminants faces important challenges. Application of semen by AI is less efficient because of the female size. Therefore, sperm quality, which is negatively affected by cryopreservation [40,55], has a critical impact. There are important differences in terms of sperm quality and fertilizing ability between fresh and frozen–thawed semen due to cold shock, ice crystal formation, membrane alterations and oxidative stress [44,54]. Membrane integrity and sperm structure can also be compro- mised by oxidative stress, resulting in lower sperm quality and fertilization rates [25,26]. Damage of spermatozoa involves the oxidative attack on the methylene groups of unsaturated phos- pholipids, leading to lipid peroxidation (LPO) [31,42]. Although semen possesses an antioxidant system, which includes glutathi- one peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and other antioxidants, their activity is affected by cryo- preservation, which increases the intensity of LPO [13,42]. There- fore, naturally occurring antioxidants may be insufficient to prevent LPO on sperm cells during the freezing–thawing process. Therefore, the addition of antioxidants to the extender may have positive effects [34,43,50,56]. There is a great variety of antioxidant substances, including vitamins, enzymes and other free radical scavengers, and their mechanism of action, toxicity and effectiveness vary enormously. Moreover, the effect of antioxidants may change depending on species, medium and protocols [37], and in some cases their presence could be detrimental [38]. http://dx.doi.org/10.1016/j.cryobiol.2014.05.004 0011-2240/Ó 2014 Elsevier Inc. All rights reserved. q Statement of funding: This manuscript was supported by University of Tabriz. ⇑ Corresponding author. Fax: +98 411 3356004. E-mail addresses: abozar.najafi@tabrizu.ac.ir (A. Najafi), hsz6955@yahoo.com, daghighkia@tabrizu.ac.ir (H.D. Kia). Cryobiology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo Please cite this article in press as: A. Najafi et al., Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.05.004