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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 combinations 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 examination. 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.

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Background Freeze‐thawing process negatively affects ram spermatozoa in terms of sperm quality, DNA integrity and antioxidant defence system. Thus, antioxidant supplementation of spermatozoa during freeze‐thawing is suggested to improve sperm parameters. Objectives The aim of this study was to determine the effects of fetuin and trehalose added into ram semen extender on sperm parameters, antioxidant parameters, antioxidant‐related gene expressions and DNA integrity during the freeze‐thawing process, in low glycerol concentration. Methods Semen samples collected from six mature rams were pooled and splitted into equal aliquots and diluted with a tris‐based extender containing different concentrations of glycerol (G5; %5 and G3; %3), fetuin (F; 2.5, 5 and 15 mg/mL) and trehalose (60 mm) as eight groups (G5F0, G5F2.5, G5F5, G5F15, G3F0, G3F2.5, G3F5 and G3F15). Results G3F5 group resulted in the highest motility, mitochondrial activity and viability and the lowest DNA fragmentation and DNA damage (p < 0.05). Also, G3F0 displayed considerably more cryoprotective effect compared with G5F0 group (p < 0.05) in terms of motility, mitochondrial activity and viability rates. Lipid peroxidation levels decreased in G5F5 group compared with G5F0 group (p < 0.05). The levels of total glutathione increased in G3F2.5 group (p < 0.05) in comparison with the G5F0 group. NQO1 gene levels were upregulated approximately twofold in G5F5, G5F15, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). The levels of GCLC gene were approximately twofold higher in G3F0, G3F2.5, G3F5 and G3F15 groups compared with G5F0 group (p < 0.05). GSTP1 gene levels were significantly higher with different levels in all treatment groups except for G5F2.5 and G3F0 groups in comparison with G5F0 group (p < 0.05). Conclusions Co‐supplementation of tris‐based extender having low glycerol (3%) with trehalose and fetuin to enhance the quality of ram spermatozoa after freeze‐thawing process is recommended.

Low temperature has been utilized to keep living cells and tissues dormant but potentially alive for cryopreservation and biobanking with great impacts on scientific and biomedical applications. However, there is a critical contradiction between the purpose of the cryopreservation and experimental findings: the cryopreserved cells and tissues can be fatally damaged by the cryopreservation process itself. Contrary to popular belief, the challenge to the life of living cells and tissues during the cryopreservation is not their ability to endure storage at cryogenic temperatures (below −190°C); rather it is the lethality associated with mass and energy transport within an intermediate zone of low temperature (−15 to −130°C) that a cell must traverse twice, once during cooling and once during warming. This chapter will focus on (1) the mechanisms of cryoinjury and cryopretection of human sperm in cryopreservation, and (2) cryopreservation techniques and methods developed based on the understanding of the above mechanisms.2 can be caused to the cells during the cryopreservation process. In this review, the mechanisms of damage to the sperm cells during the process of cryopreservation will be taken under spotlight and we will try to elucidate them in a causeeffect manner. What is cryobiology?Cryobiology is a multidisciplinary science, studying the physical and biological behaviors of living materials (e.g., cells and tissues) at low temperatures. Cryobiology contains many disciplines such as, cellular biology, theriogenology and molecular biology, engineering and mathematics, veterinary and human medicine, intensive and extensive farming on land and in watery environments [6]. Optimization of the cryopreservation procedure of spermatozoa needs all the above-mentioned disciplines because of the complex cellular structure, activation and capacitation mechanisms of spermatozoon [7].

Background: Sperm cryopreservation has been widely used in the field of reproductive biotechnology. It applies to certain males of economic and scientific values, including livestock breeds or endangered animal species. The development of a semen extender with a low cryoprotectant concentration and an appropriate amount of trehalose and boron can prevent the deterioration of sperm parameters. Objective:The main goal of this study is to establish a suitable ram extender model, by examining different combinations of high (5%) and low (3%) glycerol concentrations (to reduce its toxic effects on sperm freezing), a fixed amount of trehalose and an increased dose of boron to prevent the deterioration of sperm parameters, and investigate the levels of gene expressions. Materials and methods:The Merino ram ejaculates were collected. The collected ejaculates providing the defined criteria were pooled. The pooled ejaculates were divided into eight aliquots and diluted with the Tris extender including different combinations of glycerol (5% and 3%) and boron (0.25, 0.5, and 1 mM) concentrations and a fixed amount of trehalose, then frozen. After freeze-thawing process, sperm motility, mitochondrial membrane activity, plasma membrane integrity, acrosomal membrane integrity, DNA damage (single cell gel electrophoresis (COMET) and TUNEL assays)

In this study, the effects of diosmin and naringin on freezability of ram semen were investigated. For this purpose, 6 Merino rams were used during the breeding season. The ejaculates were pooled after being collected from the rams. Pooled ejaculates were divided into 6 groups as control (C; no additive) Naringin (1, 2, and 4 mM) and Diosmin (2 and 4 mM) groups, and then reconstituted with TRIS-based diluent. Pooled semen was equilibrated, placed in 0,25 mL straws with 10 × 107 sperm cells in each straw and frozen in liquid nitrogen vapor. After waiting for 24 hours, the straws were thawed at 37 oC for 25 s and analyzes were made. There was no statistical difference in total motility between the groups. D2 and D4 preserved the integrity of the plasma membrane better than the other groups. D4 showed better effect than other groups in terms of acrosome integrity, there was no difference between the groups in terms of mitochondrial activity. In the analysis of the sperm membrane lipid profile, it was observed that the diosmin added group had the highest lipid-phospholipid ratio. In the sperm membrane protein profile analysis, it was observed that both additives had protective effects at different levels. The highest total protein amount was seen in D4 and N4 groups. 8-OhDG positivity was more common in the control group than in the diosmin and naringin groups. Cu-Zn SOD positivity was less in the control group but more intense in all other groups. The positives were especially in the acrosome parts of the sperm cells.

In this study, the effects of diosmin and naringin on freezability of ram semen were investigated. For this purpose, 6 Merino rams were used during the breeding season. The ejaculates were pooled after being collected from the rams. Pooled ejaculates were divided into 6 groups as control (C; no additive) Naringin (1, 2, and 4 mM) and Diosmin (2 and 4 mM) groups, and then reconstituted with TRIS-based diluent. Pooled semen was equilibrated, placed in 0,25 mL straws with 10 × 107 sperm cells in each straw and frozen in liquid nitrogen vapor. After waiting for 24 hours, the straws were thawed at 37 oC for 25 s and analyzes were made. There was no statistical difference in total motility between the groups. D2 and D4 preserved the integrity of the plasma membrane better than the other groups. D4 showed better effect than other groups in terms of acrosome integrity, there was no difference between the groups in terms of mitochondrial activity. In the analysis of the sperm membrane lipid profile, it was observed that the diosmin added group had the highest lipid-phospholipid ratio. In the sperm membrane protein profile analysis, it was observed that both additives had protective effects at different levels. The highest total protein amount was seen in D4 and N4 groups. 8-OhDG positivity was more common in the control group than in the diosmin and naringin groups. Cu-Zn SOD positivity was less in the control group but more intense in all other groups. The positives were especially in the acrosome parts of the sperm cells.