Source of funding: PGCFISH (AGL2008-02172) Project, Ph.D. fellowship JAE-DOC (T. Pacchiarini) and a Ramon and Cajal Research Contract, RYC-2007-01650 (E. Cabrita). Acknowledgments Thanks to the fish farms Stolt Seafarm (Spain), A. Coelho e Castro (Portugal) and to IPIMAR for providing the fish juveniles. E-mail address: cryomedicine@aol.com (T. Kitamura) http://dx.doi.org/10.1016/j.cryobiol.2012.07.017 17. Vitrification of intact human articular cartilage. Nadr M. Jomha, Janet A.W. Elliott, Garson K. Law, Babak Maghdoori, J. Fraser Forbes, Alireza Abazari, Adetola B. Adesida, Leila Laouar, Xianpei Zhou, Locksley E. McGann, University of Alberta, Edmonton, Canada Large articular cartilage joint defects result in joint degeneration and osteoar- thritis. The only method currently able to biologically deal with this problem is osteochondral allografting but existing storage techniques are unable to maintain cell viability for long periods of time. Successful vitrification of intact articular car- tilage would allow storage of intact articular cartilage with live cells indefinitely enabling the establishment of a tissue bank that can support a joint transplantation program. We hypothesized that sequentially exposing intact human articular carti- lage to a number of cryoprotective agents (CPAs) at lowering temperatures would enable successful vitrification with high cell viability. Intact human articular cartilage on its bone base was harvested from total knee arthroplasty donors and deceased donor after ethical approval from our local institu- tion. The cartilage was exposed to four CPAs (Me 2 SO, glycerol, propylene glycol, and ethylene glycol) at lowering temperatures (0 °C, À10 °C, À15 °C) for mathematically determined times to achieve a desired concentration of each CPA at the cartilage- bone interface. After full equilibration in the final vitrification solution the samples were stored in liquid nitrogen for a minimum of 30 min and up to 3 months. The sam- ples were warmed in a 37 °C water bath and cell viability was determined using membrane integrity stains while cell functionality was documented using a mito- chondrial assay as well as pellet cultures to demonstrate collagen II and sulphated proteoglycan production. Eighteen 10 mm diameter dowels from 10 knee arthroplasty patients demon- strated 75.4 + 12.1% cell recovery as determined by membrane integrity stains. Two further samples demonstrated maximal mitochondrial function that was 95% of fresh control sample maximal function although there was a five day incubation period required prior to achieving these results. Pellet cultures demonstrated production of sulphated proteoglycans and collagen II shown by safranin O histology staining and immunohistochemistry, respectively, which was similar to fresh controls. Differ- ent sample types such as larger tissue sections, tissue from deceased human donors (both small and large sections) and one dowel stored for 3 months all demonstrated similar cell recovery as determined by membrane integrity stains. This is the first report of successful vitrification of intact human articular cartilage on its bone base – a requirement for transplantation. We have demonstrated good to excellent cell recovery as determined by membrane integrity stains. The surviving cells are functional and can produce cartilage specific macromolecules such as sulph- ated proteoglycans and collagen II indicating that these cryopreserved cells should be able to maintain the cartilage matrix over the long term after transplantation. The work in this abstract has been submitted for journal publication. Source of funding: This research was primarily funded by the Canadian Institutes for Health Research (MOP 93805 and MOP 86492). A studentship was also provided by the Alberta Heritage Foundation for Medical Research. JAWE holds a Canada Research Chair in Thermodynamics. Some authors have filed a patent related to this technique. E-mail address: njomha@ualberta.ca (N.M. Jomha) http://dx.doi.org/10.1016/j.cryobiol.2012.07.018 18. Vitrification of zebrafish (Danio rerio) ovarian follicles. Leandro Cesar Godoy 1,2 , Tiziana Zampolla 2 , Danilo Streit Jr. 1 , Adriana Bos-Mikich 1 , Tiantian Zhang 3 , 1 Aquam Research Group, Federal University of Rio Grande do Sul, Brazil, 2 IBEST, University of Bedfordshire, UK, 3 School of Applied Sciences, Bournemouth University, UK Overfishing and environmental factors such as pollution have contributed to the significant decline of fish populations worldwide. Successful cryopreservation of fish gametes and embryos would have important applications in conservation and aqua- culture. Although cryopreservation of fish semen has already been achieved success- fully, cryopreservation of fish embryos and oocytes remains elusive. Despite the widespread use of vitrification for oocyte cryopreservation in humans and domestic mammals, vitrification of fish oocytes has not been studied to date. This study aimed at developing a cryopreservation protocol for stage III zebrafish ovarian follicles within fragment using vitrification. Vitrification ability of methanol, ethanol, Me 2 SO, propylene glycol and ethylene glycol were tested using a range of concentrations made up in 90% Leibovitz L-15 medium. Solutions were tested for vitrification ability using 0.25 ml plastic straw and a fibreplug TM (CryoLogic Ltd.) loaded by a pipette. The transparent glassy appearance during cooling and warming was used to identify vit- rified solution. Based on results obtained from this stage, the vitrification ability of 24 combinations using the mentioned cryoprotectants was studied. Three vitrifica- tion solutions (VS) (V6B: 1.5 M methanol + 6.0 M ethylene glycol + 0.5 M sucrose; V1B: 1.5 M methanol + 4.5 M propylene glycol; and V2: 1.5 M methanol + 5.5 M Me 2 SO) were selected for subsequent experiments based on their vitrifying ability. For VS toxicity tests, ovarian follicles were equilibrated in L-15 medium containing 1.5 M methanol for 30 min at room temperature. After equilibration follicles were exposed to VS for 3 min in a stepwise manner. Afterwards the cryoprotectants were gradually removed in three steps and viability was assessed by trypan blue staining. For vitrification procedures, immediately after exposure to VS ovarian follicles were loaded in either plastic straw or fibreplug TM and plunged into liquid nitrogen, being stored for at least 20 min. The warming was performed at 28 °C and cryoprotectants were gradually removed in three steps. Ovarian follicle viability was assessed by three means: trypan blue staining, measurement of ATP level in the follicles and assessment of mitochondrial membrane potential and distribution by using JC-1 molecular probe. Results obtained from toxicity tests showed follicles viability of 74% for V1B; 30% for V2 and 78% for V6B. After vitrification, ovarian follicles pre- sented a viability of 60% when fibreplug and V1B were used, V2 showed viability of 42% (using fibreplug), and using plastic straws V6B solution showed only 2% of viability. [ATP] soon after warming dropped down to 22% (V2) and 7% (V1B) and two hr later the concentration in V2 was 15% and for V1B a very low concentration of 3.5%. The viable follicles in V1B and V2 seemed to be opaque and morphologically intact, when assessed immediately after warming. These results are different to the results described so far in the literature, where zebrafish follicles were observed to become swollen and translucent even during the warming process after being exposed to controlled slow cooling protocols. However, 30 min after warming most of the follicles started to become semi-translucent and lightly swollen. A complete loss of internal structure pattern and also loss of fluorescence indicated follicles with no mitochondrial membrane potential after vitrification. Conflict of interest: None declared. Source of funding: L.C. Godoy has been awarded a Ph.D. fellowship from the CAPES Foundation – Brazilian Ministry of Education. This research has been funded by the iBEST strategic research fund. E-mail address: godoyaqua@yahoo.com.br (L.C. Godoy) http://dx.doi.org/10.1016/j.cryobiol.2012.07.019 19. Cryopreservation and fertility of geoduck (Panopea zelandica) sperm and oocytes. Serean L. Adams, J.F. Smith, H. Robin Tervit, Samantha L. Gale, Lindsay T. McGowan, Jonathon R. Morrish, E. Watts, J. Taylor, Cawthron Institute, Private Bag 2, Nelson, New Zealand 7042. AgResearch, Private Bag 3123, Ruakura, Hamilton 3240, New Zealand Geoducks are large salt water clams. They are currently farmed in North Amer- ica. In New Zealand, there is a small wild fishery for the native geoduck, Panopea zelandica. More recently, there is increasing interest in farming this high value spe- cies in New Zealand. Already a small batch has been reared and is ready to be exper- imentally on-grown on existing mussel farms. Cryopreservation can be a powerful tool for hatcheries and selective breeding programmes for aquaculture. In hatcheries, cryopreservation allows year-round spat supply without the need to condition broodstock for out of season production. This is particularly useful with new species where optimized conditioning regimes have not been established. Spawning techniques may also need development and cryopreser- vation can enable hatcheries to capitalize on single sex spawnings where gametes would otherwise be wasted. In selective breeding, cryopreservation enables breeders to make crosses on demand; providing them with complete flexibility and enabling faster genetic gains. Towards this goal, we investigated cryopreservation and fertility of gametes from the New Zealand geoduck, Panopea zelandica. Geoducks were induced to spawn dur- ing their natural spawning season using thermal shock and serotonin injection. Sperm was collected as concentrated as possible by aspiration and stored at 4 °C before being used in experiments. Oocytes were maintained at either room temperature or 4 °C. For both sperm and oocytes, three cryoprotectants, ethylene glycol, dimethyl sulphoxide and propylene glycol were made up in Milli-Q water and evaluated at final concentrations of 10% and 15% with or without 0.2 M trehalose. For sperm, a simple freezing method utilizing a rack attached to a polystyrene frame which floated the rack 3 cm above the liquid nitrogen surface was evaluated as a method of freezing. Sperm were diluted 1:1 with cryoprotectant solutions and loaded into 0.5 mL straws 344 Abstracts / Cryobiology 65 (2012) 339–366