Cryoprotective Effect of Vitamin E Supplementation of Different Extenders on Quality and Fertilizing Ability of Frozen-Thawed Brown Trout Sperm Yusuf Bozkurt, 1 _ Ilker Yavas x, 2 Mustafa Numan Bucak, 3 Tug ˘ ba Raika Kıran, 4, * and Aziz Gu ¨l 5 Vitamin E is one of the most powerful antioxidants for prevention of cell damage resulting from cryopreser- vation, but its efficacy for cryopreserving brown trout sperm is still unclear. In this work, the protective effect of vitamin E on quality, fertilizing capacity, and DNA damage of brown trout (Salmo trutta macrostigma) sperm after cryopreservation was evaluated. Sperm samples were diluted at the ratio of 1:10 with three different extenders (E): (E-I): 300 mM glucose, 10% egg yolk; (E-II): 33.3 mM glucose, 5.1 mM NaCl, 0.5 mM NaH- CO 3, , 15% DMA; and (E-III): 61.6 mM NaCl, 134.2 mM KCl, 1.9 mM CaCl 2 , 0.8 mM MgCl 2 , 2.3 mM NaHCO 3 in distilled water. Each extender was supplemented with 10% DMSO and different concentrations of vitamin E at 0.1, 0.5, and 1.0 mM. Spermatozoa frozen without vitamin E (0 mM, control) and fresh sperm were also used. After dilution, the sperm was aspirated into 0.25 mL straws, frozen 3 cm above the liquid nitrogen (LN 2 ) surface, and plunged into the LN 2 . Cell motility, viability, fertilization, and eyeing were determined in post- thawed samples. DNA damage was determined by the comet assay after cryopreservation. Supplementation of 1 mM vitamin E to all extenders exhibited the best cryoprotective effect in terms of sperm motility, duration of motility, viability, fertility, and DNA integrity against cryopreservation damage, compared with 0.1, 0.5, and control group (0 mM) ( p < 0.05). The highest post-thaw motility (62.4% – 0.36%), fertilization (48.2 – 0.84), and the lowest DNA damage (7.245%) were obtained with the extender-II including 1.0 mM vitamin E ( p < 0.05). Consequently, vitamin E positively affected the motility parameters, fertility, and DNA integrity, and the results suggest the addition of extenders with vitamin E as an antioxidant for the cryopreservation of brown trout sperm. Keywords: vitamin E, antioxidant, brown trout, sperm quality, DNA damage, cryopreservation Introduction C ryopreservation is a long-term storage technique that is efficiently used as an important biotechnological tool for the aquaculture industry. Its benefits can be summa- rized as follows: synchronization of the availability of the gamete year-round, reduction of the nursing cost of brood- stock, promotion of breeding techniques for broodstock man- agement, and development of the hatchery management by protecting sperm of the selected individuals in cryobanks. 1,2 Thus, the improvement of effective cryopreservation methods is very important to facilitate the use of cryopreserved sperm for large-scale larvae production. Salmo trutta macrostigma is one of the important fresh- water fish species in terms of biodiversity and its econom- ical value. Unfortunately, its population declines day by day because of illegal practices and heavy fishing pressure, degradation of spawning habitats, and river damming. 3,4 Cryopreservation of salmonid sperm has been widely investigated because of its high commercial and recreational value in terms of the food sector and fishing. On the con- trary, the sperm cryopreservation process, despite its various 1 Department of Aquaculture, Faculty of Marine Sciences and Technology, _ Iskenderun Technical University, _ Iskenderun, Turkey. 2 Deparment of Reproduction and Insemination, Faculty of Veterinary Science, Mustafa Kemal University, Antakya, Turkey. 3 Deparment of Reproduction and Artificial Insemination, Faculty of Veterinary Science, Selc ¸uk University, Konya, Turkey. 4 Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences, _ Iskenderun Technical University, _ Iskenderun, Turkey. 5 Department of Animal Science, Faculty of Agriculture, Mustafa Kemal University, Antakya, Turkey. *Present affiliation: Malatya Turgut O ¨ zal University, Health Services Vocational School, Medical Labratory Techniques Programme, Malatya, Turkey. BIOPRESERVATION AND BIOBANKING Volume 19, Number 3, 2021 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2020.0062 171 Downloaded by 3.87.19.40 from www.liebertpub.com at 04/02/22. For personal use only.