Cryopreservation Effects on Ram Sperm Ultrastructure Nazan Keskin, 1 Cennet Erdogan, 2 Mustafa Numan Bucak, 3 Ali Erdem Ozturk, 3 Mustafa Bodu, 3 Pınar Ili, 4 Nuri Baspinar, 5 and Sukru Dursun 6 Cryoprotectants are known to have protective effects against cryodamage to spermatozoa. In this study, the cryoprotective effects of two cryoprotectants (glycerol, ethylene glycol) and cryoprotectants/trehalose combi- nations on frozen-thawed ram spermatozoa were investigated at the ultrastructural level. For this purpose, ejaculates collected from Konya Merino rams were pooled and diluted with a tris-based extender containing additives, including 5% glycerol, 3% glycerol +60 mM trehalose, 1.5% glycerol +100 mM trehalose, 5% ethylene glycol, 3% ethylene glycol +60 mM trehalose, and 1.5% ethylene glycol +100 mM trehalose. They were all cooled to 5°C and then frozen in 0.25 mL French straws in liquid nitrogen. The samples were thawed at 37°C and centrifuged to remove the diluents. Then, they were processed using a scanning transmission electron microscope. In the statistical analysis, the number of ultrastructurally cryodamaged and intact spermatozoa were counted in longitudinal and transverse ultrathin sections in all groups by electron microscopic exami- nation. The amount of intact spermatozoa in the groups containing 5% ethylene glycol and 1.5% ethylene glycol +100 mM trehalose was found to be higher than other groups ( p < 0.05). As a result, it was suggested that the groups of 5% ethylene glycol and 1.5% ethylene glycol +100 mM trehalose provided the highest protection for the ultrastructural morphology of frozen-thawed Konya Merino ram spermatozoa among the groups. Keywords: cryopreservation, Konya Merino ram spermatozoa, trehalose, ethylene glycol, glycerol, electron microscopy Introduction S perm cryopreservation has recently become widely used in reproductive biotechnology to maintain fertil- ity. However, sperm cryodamage involves cellular dam- age, including changes in the membranes, mitochondria, acrosomes, and axonemes. 1–5 Such cellular cryodamage adversely affects spermatozoa fertility by decreasing the viability of the cells. Numerous studies have been conducted on the role of various cryoprotectants in cell cryopreservation. 6–10 Low-molecular- weight cryoprotectants may prove to allow less damage to the spermatozoa. 11 On the one hand, it was reported that ethylene glycol re- duced abnormality and improved sperm viability, acrosome integrity, plasma membrane integrity, conception rate, and pregnancy rate of bull semen compared to glycerol. 12 In another study, Awad 13 compared the cryopreservation effects of low-molecular-weight cryoprotectants (ethylene glycol) to glycerol on post-thawed computer-assisted semen analysis (CASA) sperm parameter in bull semen. They found no ad- vantage to using ethylene glycol to replace glycerol in bull semen freezing. However, they reported that the possibility of using ethylene glycol as a permeating cryoprotectant for bull semen deserved further investigation, and these cryoprotec- tants should also be evaluated in extenders that contain di- saccharides or cholesterol. Trehalose is a naturally occurring sugar containing two d-glucose units in an a,a-1,1 linkage. It can protect proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions, including desic- cation, dehydration, heat, cold, and oxidation. 14 It has been concluded that the antioxidant properties of the extender trehalose may be related to its effectiveness in membrane cryopreservation. 15–19 The cryoprotective effects of treha- lose may be due to enhanced sperm membrane fluidity before freezing. 20 In addition, it preserves the structural integrity of the cells and protects them against freezing- 1 Department of Histology and Embryology, Pamukkale University Faculty of Medicine, Denizli, Turkey. 2 Department of Histology and Embryology, Pamukkale University Graduate School of Health Sciences, Denizli, Turkey. 3 Department of Reproduction and Artificial Insemination, Selcuk University, Veterinary Faculty, Konya, Turkey. 4 Denizli Health Services Vocational High School, Pamukkale University, Denizli, Turkey. 5 Department of Biochemistry, Selcuk University, Veterinary Faculty, Konya, Turkey. 6 Department of Gynecology and Obstetrics, Aksaray University, Veterinary Faculty, Aksaray, Turkey. BIOPRESERVATION AND BIOBANKING Volume 18, Number 5, 2020 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2020.0056 441 Downloaded by 54.161.150.205 from www.liebertpub.com at 11/04/21. For personal use only.