Andrologia. 2019;00:e13393. wileyonlinelibrary.com/journal/and | 1 of 6 https://doi.org/10.1111/and.13393 © 2019 Blackwell Verlag GmbH 1 | INTRODUCTION Semen cryopreservation and artificial insemination can be used to control spread of many infectious diseases and promote dissemi‐ nation of superior genetics (Dalal, Ajeet, Ravi, Gyan, & Chandolia, 2018). However, utilisation of this assisted reproductive technology depends on quality of frozen‐thawed semen, which can be influ‐ enced by many factors, including sperm cryoresistance (Inanc, Tekin, et al., 2018). For example, changes in lipid composition of sperm membranes affect quality of frozen‐thawed spermatozoa (Amorim, Graham, Spizziri, Meyers, & Torres, 2009). Novel freezing methods and semen extenders with various supplements are still being investigated to further improve quality of cryopreserved ram spermatozoa (Blackburn, 2004). Adding an‐ tioxidants to extenders for chilling or freezing semen may confer antioxidant protection and improve sperm motility, mitochondrial activity and viability (Bucak, Coyan, Ozturk, Gungor, & Omur, 2012). Lipid composition (lipid/cholesterol ratio) also affects abil‐ ity of spermatozoa to capacitate and undergo an acrosome reac‐ tion (He, Bailey, & Buhr, 2001; Moore, Squires, & Graham, 2005). Specifically, ram spermatozoa are sensitive to cryopreservation‐ induced damage, due to their low phospholipid/cholesterol ratio (Inanc, Uysal, & Ata, 2018). Furthermore, cryopreserved sperma‐ tozoa are more sensitive to oxidation than fresh spermatozoa, due to less effective intracellular antioxidant defence mechanisms (Chandra et al., 2012). Received: 8 May 2019 | Revised: 18 July 2019 | Accepted: 19 July 2019 DOI: 10.1111/and.13393 ORIGINAL ARTICLE Gallic and carnosic acids improve quality of frozen‐thawed ram spermatozoa Sukru Gungor 1 | Muhammed E. Inanc 1 | Caner Ozturk 2 | Firat Korkmaz 3 | Ilktan Bastan 3 | Beste Cil 4 | John P. Kastelic 5 1 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University, Burdur, Turkey 2 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Turkey 3 International Center for Livestock Research and Training, Ankara, Turkey 4 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey 5 Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada Correspondence Sukru Gungor, Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University, 15030, Burdur, Turkey. Email: sukrugungor85@gmail.com Abstract The objective was to determine effects of gallic acid (GA) and carnosic acid (CA), present in carob pods and rosemary extract respectively, on frozen‐thawed ram sper‐ matozoa. Thirty ejaculates were collected from five Merino rams, pooled, diluted in Tris‐based extender and divided into five equal portions containing: 0.05 or 2 mM of GA; 0.05 or 0.2 mM of CA; or no additive (control). Extended semen was equilibrated at +4°C, loaded into straws, held 5 cm above liquid nitrogen for 12 min then plunged. Computer‐aided sperm analysis was used to assess motility, whereas flow cytometry was used to assess high mitochondrial membrane potential (HMMP) and percentages of spermatozoa with plasma membrane and acrosome integrity (PMAI). Spermatozoa supplemented with 2 mM GA had greater total motility than control spermatozoa (39.9 ± 3.01 vs. 29.2 ± 1.31%, mean ± SEM, p < .05). The PMAI was greatest in 0.2 mM CA (13.3 ± 0.68%), whereas HMMP was highest in 0.05 mM CA but lowest in control (22.9 ± 4.95 and 11.4 ± 3.64% respectively; p < .05). In conclusion, for cryopreserva‐ tion of ram semen in Tris‐based extender, supplementation with 2 mM GA increased post‐thaw motility, whereas supplementation with 0.05 mM CA enhanced mitochon‐ drial function. KEYWORDS carnosic acid, computer‐assisted sperm analysis, flow cytometry, gallic acid