Chilled storage of semen from Atlantic halibut, Hippoglossus hippoglossus L. I: Optimizing the protocol Igor Babiak a, * , Oddvar Ottesen a , Geir Rudolfsen b , Steinar Johnsen a a Bodø University College, Department of Fisheries and Natural Sciences, 8049 Bodø, Norway b University of Tromsø, Department of Biology, 9037 Tromsø, Norway Received 5 October 2005; received in revised form 9 June 2006; accepted 9 June 2006 Abstract Asynchrony in gamete production between females and males, and decrease in semen quality towards the end of reproductive season make chilled short-term storage of Atlantic halibut, Hippoglossus hippoglossus, semen a desirable method to apply for artificial propagation of this fish species. The goal of the present study was to determine the critical physiochemical factors that affect the success of chilled storage of halibut spermatozoa, and to develop a reliable, simple and efficient protocol for the storage. The presence and type of gaseous atmosphere, dilution and type of diluent, dilution ratio, and additional factors including spermatozoa sedimentation and replenishing the storage medium were tested in relation to spermatozoa motility parameters. Also, fertilization tests were performed with stored semen. Normoxia (air atmosphere) conditions were superior to both hyperoxia (pure oxygen) and no gaseous atmosphere for chilled storage. Dilution of semen with a diluent was superior to incubating undiluted semen. A dilution factor of between 6 and 10 times the original semen volume resulted in the longest viability of stored spermatozoa. Preventing spermatozoa sedimentation through daily swirling of the samples was superior to weekly swirling, however the effect was negligible for the first month of storage. Replenishing the storage medium showed no advantage to incubating in unchanged medium. Semen diluted in modified Hanks’ balanced salt solution 1:5–1:9, supplemented with antibiotics, and kept at 0–1 8C in Ziploc bag filled with air retained its viability for exceptionally long time. A decrease in the percentage of motile spermatozoa was observed after 43 days of storage, and a decrease in curvilinear velocity occurred after 15 days. Samples remained motile for at least 79 days of storage and the fertilization ability was retained for at least 70 days of storage. The results demonstrate a high potential of application of chilled storage of semen into reproduction programs in Atlantic halibut aquaculture. # 2006 Elsevier Inc. All rights reserved. Keywords: Atlantic halibut; CASA; Preservation; Fish; Sperm 1. Introduction The Atlantic halibut, Hippoglossus hippoglossus L., is the world’s largest flatfish, and because of its high market value is regarded as a coldwater marine species having a very high aquaculture potential. Halibut breed normally between January and April [1]. Males produce high quantities of semen, but its quality decreases towards the end of reproductive season, mostly because of dehydration and ageing processes to spermatozoa [2–5]. The presence of low quality of sperm at the end of reproductive season, at a time when high quality eggs are still being produced by females, represents a major problem in halibut aquaculture as it restricts the ability www.journals.elsevierhealth.com/periodicals/the Theriogenology 66 (2006) 2025–2035 * Corresponding author. Tel.: +47 75517922; fax: +47 75517349. E-mail address: Igor.Babiak@hibo.no (I. Babiak). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.06.003