Bas ¸ tan 4 . 1 Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology, Afyon Kocatepe University, Turkey; 2 Histology and Embryology Department, School of Medicine, Ankara University, Turkey; 3 Faculty of Pharmacy, Department of Pharmacology, Laboratory Animal Care and Research Unit, Gazi University, Turkey; 4 Faculty of Veterinary Medicine, Department of Obstetrics and Gynecology, Ankara University, Turkey * Corresponding author. E-mail address: dbakiacar@gmail.com (D.B. Acar). The vitrification of ovarian tissue has the potential to preserve the fertility. The purpose of the present study was to reveal the effects of cryoprotec- tant agent temperature on the morphological and ultrastructural features of follicle in dog ovarian tissue. The ovarian tissues were classified as control and two vitrification groups. The forty tissues were exposed to cryoprotectants at 4  C (Group I) or room temperature (RT) (Group II). To vitrification procedure, 1 mm 3 pieces of ovarian tissue were placed in an acupuncture needle. The tissues were exposed to 7.5% ethylene glycol and 7.5% dimethyl sulfoxide (Me 2 SO 4 ) for 10 min and then 15% ethylene glycol and 15% Me 2 SO 4 for 2 min, either at room temperature or at 4  C and stored in liquid nitrogen at least 1 week. After then the tissues were thawed and immersed into Bouins' and glutaraldehyde solution for analysis. The tis- sues which were vitrified at 4  C were more preserved than at the room temperature. Lots of healthy primordial follicles were observed in the Group I. In Group II, there were very few normal follicles. In the ultra- structural examination in the Group I, it was observed that the double membrane structure of the oocytes’ nucleus was preserved, the distribu- tions and structures of the organelles were normal. In Group II ultra- structural observation, the oocyte cytoplasm was damaged and the linkages between oocytes and the follicle cells were detached. The ice damage was noticed in the tissue structure in this group. Oocytes vacuoles and interstitial vacuoles were seen. The optimal agent temperature was determined as 4  C for dog vitrification equilibration. Statement of Funding: Not applicable Conflict of Interest: None to disclose P74 MOTILITY AND MEMBRANE FUNCTIONALITY OF POST-THAW BULL SPERM UNDER EFFECTS OF LOW CONCENTRATIONS OF HYDROGEN PEROXIDE Fatemeh Khosrozadeh 1 , Amir Karimi 1 , Mohsen Sharafi 2, * . 1 Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran; 2 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran * Corresponding author. E-mail address: m.sharafi@royaninstitye.org (M. Sharafi). Sperm freezing is an important method that is widely used for long-term storage of sperm. But during the freezing process, the sperm is subjected to biochemical, osmotic, physical, mechanical and oxidative stress. Free radicals such as reactive oxygen species (ROS), are one of the causes of cryo- damages that can adversely affects the sperm performance. However, in recent years, very low concentration of ROS could improve the quality of post-thawed sperm in certain mammalians. The aim of this study was to consider the effects of very low concentration of hydrogen peroxide on parameters of motility and membrane integrity after freezing process. Motility and membrane integrity were assessed using computer aided sperm analysis (CASA) and Hypoosmotic swelling test, respectively. Semen sample in fresh group, control freezing group and a freezing group con- taining very low concentration of hydrogen peroxide were assessed (18 samples from 3 bulls). The results of the study showed that applying very low concentration of hydrogen peroxide improves the motility of bull sperm after thawing.There was no significant difference in membrane integrity between the fresh, control freezing and treatment freezing groups. It is well-known that ROS has a dual role in the biology of sperm and can improve the cryo-tolerance of bull sperm if the very low con- centration was used. Source of Funding: This work was supported by funding from Royan Institute and Tabriz University. Conflict of Interest: None to disclose P75 MOTION CHARACTERISTICS AND MITOCHONDRIAL MEMBRANE POTENTIAL OF BULL SPERM CRYOPRESERVED IN HOME-MADE SOYBEAN BASED EXTENDER Razieh Fouladvandy 1 , Ali Akbar Masoudi 1 , Mohsen Sharafi 2, * . 1 Department of Animal Science, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran; 2 Department of Poultry Science, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran * Corresponding author. E-mail address: m.sharafi@modares.ac.ir (M. Sharafi). Cryopreservation has been applied as a routine technique for processing bull sperm for artificial insemination. However, around 30-40 % of the thawed sperm are not viable. Therefore, the use of an optimal extender for cryopreservation of sperm appears to be necessary. This study assessed the effect a home-made extender containing soybean lecithin for cryopreser- vation of bull sperm (18 samples from 3 bulls). Motion characteristics and mitochondrial membrane potential of bull sperm were assessed after thawing. The indicators of motion characteristics were assessed using computer aided sperm analysis (CASA) and mitochondrial membrane potential using the JC1 probe. Data were analyzed using general linear model procedure using SAS 9.1 and Tukey’s test was used to determine statistical differences among groups (P0.05). Result showed that there was no significant difference between sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) between soybeans based extender and control extender. But mitochondrial membrane potential was higher when soy- bean lecithin-based extender was used. Beneficial effects of soybean lecithin in the extenders for cryopreservation of bull semen need to further investigated. Source of Funding: This work was supported by funding from the Tarbiat Modares University. Conflict of Interest: None to disclose P76 PROTECTION AGAINST CELL DEATH DURING CRYOPRESERVATION WITH WHEAT PROTEINS: WHICH MODE OF CELL DEATH? M elanie Chow-Shi-Y ee * , M elanie Grondi, Diana Alison Averill- Bates, François Ouellet. University of Qu ebec at Montreal, Canada * Corresponding author. E-mail address: melanie.csy@gmail.com (M. Chow-Shi-Y ee). Hepatocytes are a good physiological model for pharmaceutical and clin- ical domains. They are important for drug toxicity testing and are also useful for human cell transplantation as an alternative to liver organ transplantation. Freshly isolated hepatocytes are used immediately or stored by cryopreservation for later use. This approach requires cryopro- tective chemicals to avoid damage caused by the freeze / thaw process and prevent loss of viability and metabolic functions. One of the most commonly used cryoprotective agents is Me 2 SO, which is cytotoxic and decreases viability, metabolic functions and attachment efficiency. Our goal is to develop new cryoprotective agents that are less toxic, based on nature’s freezing tolerance-associated proteins that are present in wheat. These proteins contribute to the wheat plants’ capacity to withstand cold and freezing conditions. Two recombinant wheat-derived proteins, enolase and 2-cys peroxiredoxin, were used as cryoprotectants and improved hepatocyte post-cryopreservation viability and metabolic func- tionality, when compared to cells cryopreserved with Me 2 SO. This study investigates mechanisms of cell death induced during cryopreservation with plant proteins compared to Me 2 SO. While levels of reactive oxygen species (ROS) increased during cryopreservation with Me 2 SO, this did not occur when hepatocytes were cryopreserved with the recombinant plant proteins. These proteins therefore appear to play an antioxidant role. Given that ROS are activators of apoptosis, we investigated whether different apoptotic pathways were activated in hepatocytes during cryopreserva- tion. The principal type of cell death that occurred was apoptosis, although some necrosis occurred. Me 2 SO caused considerably more apoptosis, whereas the plant proteins provided protection. These results improve our understanding of how cells die during cryopreservation and should help to optimize cryopreservation protocols to limit loss of cell viability and Abstracts / Cryobiology 85 (2018) 120e190 189