Cryopreservation of gametes and embryos is crucial in fertility treatment and fertility preservation. Preservation of oocytes is a more complicated process than preservation of sperm and embryos. According to recent studies, a new preservation technique called vitrification is found to have higher rates of oocytes-survival after warming    The aim of this study was to evaluate VitriBlast™ Kit, for vitrification of oocytes. Vitrification is an ultra-rapid freezing method in the presence with of high concentrations of cryoprotectants which avoids intracellular ice-crystal formation during the freezing and warming process.     In this study, a total number of 117 immature oocytes were used and 62 of these oocytes were vitrified with VitroBlast™ Kit. During this process two different vitrification devices were used, VitroLoop™ and Cryopette®. After warming, the average survival rate for vitrified oocytes was found to be 61% for VitroLoop™ and 15% for Cryopette® (p<0.001). The remaining 55 oocytes were used as a non-frozen control group and the same incubation method as for vitrified oocytes was used. The survival rate for the control groups was higher than for the vitrified groups (93% versus 35%, p<0.001). The results of this study indicate that VitriBlast™ Kit is not suitable for vitrification of oocytes.

The overall aim of this thesis was to determine if day old turkey gonads could be cryopreserved and transplanted into recipient poults. This would allow for grafts to develop and mature normally and potentially produce donor-derived offspring. In addition, the monitoring of folliculogenesis in chickens was studied to determine if ultrasonography would be a useful technique to study this biological process, with the intention of using this method in future studies on ovarian graft development. Three studies were conducted: cell and tissue viability of vitrified day old turkey gonads, transplantation of day old turkey gonads into recipient poults, and monitoring of follicle growth in chickens using ultrasonography. The objective of the first study was to determine if day old turkey gonads were viable after vitrification using a standard protocol or with variation in equilibrium solution (ES) and/or vitrification solution (VS) absorption times. Three different ES time points were tested 10, 15 and 20 min (10ES, 15ES and 20ES) and two different VS time points 2 and 3 min (2VS and 3 VS). The cell and tissue viability was determined by Trypan Blue Assay and light microscopy, respectively. Testicular cell viability was conducted using three vitrification protocols and fresh tissue. All vitrification protocols along with fresh tissue were assessed by light microscopy, to evaluate histological alterations, in ovarian and testicular tissue. Protocols with the highest cell viability and best morphological scores were selected as being the most suitable for cryopreservation. Testicular tissue vitrified using 15ES or 20ES with 3VS had the highest cell viability. Ovarian and testicular tissue vitrified using 15ES with (3VS or 2VS), showed the best morphological scores, out of all the protocols. The second study was broken down into two parts: Part A (Trial 1 & 2) was to determine the most suitable age group for poults pre-surgery to give the highest survivability; Part B (Trial 3) was to determine if previously vitrified day old turkey gonads could develop and mature normally, by retrieving grafts post-surgery, at- different time points. In Trial 1, large white turkeys (LWT) 1, 3, 4 and 7 days of age were used and for Trial 2, LWT’s aged 1, 3, 4, and 5 days of age were used. In Trial 3, bronze turkeys at 1 day of age were used, and graft tissue was used from day old LWT’s previously vitrified (10ES/2VS) or fresh. For all Trials, the survivability at each time point was analyzed, and for the third Trial, the grafts recovered were histologically analysed. From Trials 1 and 2, seven and three day old poults had the highest survivability ratios (3/5 and 6/8) respectively. For Trial 3, day old male poults (96%) had a higher survivability than the females (68%). From Trial 3, transplants were only recovered in females and males up until 4 days and 4 weeks post-surgery respectively, with no fresh tissue grafts recovered. The histological analysis of testicular transplants showed deteriorating structure, with a steady progression away from normal morphology, post-surgery. The third study’s objective was to determine the growth rates and patterns of folliculogenesis in Barred Plymouth Rock (BPR) hens by using ultrasonography. Two ultrasound Trials were performed: the first to determine the optimum time interval between serial ultrasound scans to accurately map follicles, and the second to tackle the main objective of the third part of this thesis. For the first Trial, BPR hens were scanned periodically over 24 hours, follicle diameter and position were recorded and mapped with respect to the ovary and neighbouring follicles. Proportional follicle growth, compared to the first scanning session showed that the 24hr time point had the only significant (P<0.001) proportional follicle growth. It is recommended here that scans occur twice a day (morning and afternoon) to capture a more precise growth rate of follicles. In the second Trial, BPR hens were scanned twice a day, over an 11-day period. Follicle diameters (height and width) were recorded to calculate follicle area. The growth of each follicle’s area was compared to the time before ovulation to determine the overall follicle growth rate. Additionally, it was determined if time (night, day) and type of preovulatory follicle (F1-F5) played a significant role in follicle growth rates. The overall follicle growth rate was best described by a cubic equation (R2=0.907, P<0.001). Follicle growth rates were influenced by both time (P=0.009) and type (P<0.001). With F2 and F3 follicles (P<0.05) having a higher growth rate than F1 and F5 types. In conclusion, modifications to the standard vitrification protocol used on quail gonads were necessary to increase cell viability and lower morphological alterations for turkey gonads left whole. For future work it has been shown that day old turkey poults can survive gonad transplantation. The lack of development of grafts is most likely due to a combination of tissue damage after vitrification-warming procedures and insufficient immunosuppression of the host. This work has paved the way for the poultry industry to be able to cryogenically store turkey gonads and revive lines when required. Additionally it was shown that serial ultrasound scans twice a day provided accurate monitoring of follicle growth in Barred Plymouth Rock hens. For BPR hen’s follicle growth rates and patterns were successfully measured. This gives the industry another tool to better select superior laying hens and to create a more homogeneous laying flock. Future application of ultrasonography on gonad monitoring has the potential to show growth and maturation of grafted tissue before the production of donor-derived offspring, enabling earlier detection of successful transplantation.

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Cette thèse porte d'une part sur la conception et la caractérisation d'alliages métalliques pour des applications dans les domaines de la réfrigération magnétocalorique ou thermoélectrique ; ces alliages ont été conçus en tenant compte à la fois des critères dictés par l'application recherchée et des critères dictés par la nécessité de vitrifier l'alliage. Dans un deuxième temps, ce travail montre qu'il est en partie possible de prédire le domaine et l'aptitude de vitrification d'un système donné par une approche thermocinétique basée sur des données thermodynamiques.

Après dissolution du combustible et séparation des différents éléments par le procédé PUREX, la majeure partie des produits de fission et des actinides mineurs est calcinée puis vitrifiée dans des verres de conditionnement des déchets nucléaires. Parmi ces produits de fission, certains précipitent et ne sont pas immobilisés dans la phase vitreuse dédiée aux déchets de haute activité à vie longue. Les éléments platinoïdes Pd-Rh-Ru sont insolubles dans le verre nucléaire. En fonction du potentiel d'oxygène imposé par la fritte de verre, ils précipitent sous la forme de phases oxydes complexes ou de composés intermétalliques principalement alliés aux éléments chalcogènes Te et Se. Au contraire, le molybdène reste oxydé lors des dernières étapes du procédé de vitrification. Très réactif vis-à-vis des oxydes constitutifs de la fonte verrière, il forme majoritairement des molybdates. Au cours de cette thèse, la thermodynamique des systèmes chimiques contenant le molybdène (Mo), les éléments platinoïdes Pd-Rh-Ru et les chalcogènes Se-Te ont été étudiés expérimentalement. En parallèle, la thermodynamique de ces systèmes chimiques est également modélisée par la méthode Calphad. L'objectif de cette modélisation est de prédire les phénomènes de cristallisation du molybdène et des platinoïdes observés au cours des étapes de vitrification en fonction de la composition et de la température. Ces modélisations permettent d'effectuer des calculs d'applications en lien avec le procédé industriel de vitrification. / After the dissolution of the used fuel and the separation of several elements by the PUREX process, the high level nuclear wastes composed of fission products and minor actinides are reprocessed and vitrified in nuclear glasses at AREVA La Hague plant. Some of the fission products precipitate : they are not solubilized in the glass matrix. On the one hand, platinoids Pd-Ru-Rh are not soluble in the nuclear glasses. Depending on the oxygen potential, they form complex solid oxyde phases or intermetallic compounds containing chalcogen elements such as selenium and tellurium. On the other hand, the molybdenum forms only oxide phases during the vitrification process. It reacts strongly with the oxide phases present in the glass melt to form mainly molybdate phases. Some of these phases are only temporary formed but other are more stable and can precipitate in the glass matrix when a large amount of molybdenum is supplied. In this thesis, the thermodynamics of the chemical systems containing molybdenum, the platinoid elements Pd-Rh-Ru and the chalcogen elements Se and Te were experimentally investigated. At the same time, these chemical systems were modeled with the Calphad method so as to be able to predict the crystallization phenomena of molybdenum and the platinoids occurring during the vitrification as a function of the composition and the temperature. These modelings are useful to perform application calculations in relation with the vitrification process.

Cryopreservation is the preservation of biomaterials at extremely low temperatures. It is the only alternative for long-term storage of high quality biomaterials, with applications to biobanking and transplant medicine. Cryopreservation success revolves around the control of ice formation, which is known to be harmful. Ice formation is a path-dependent phenomenon, affected by the thermal history and presence of nucleation promotors. Cryoprotective agents (CPAs) are commonly added to the biomaterial to be preserved, in order to suppress ice formation and inhibit its growth during the cryopreservation protocol. Ice-free cryopreservation can be achieved in large-size systems when the biomaterial is loaded with a high CPA concentration solution and cooled rapidly, in a process that is known as vitrification (vitreous means glassy in Latin). During vitrification, the CPA viscosity increases exponentially with decreasing temperature, while the material is cooled to deep cryogenic temperatures faster than the typical time scale for crystallization. The material can potentially be stored indefinitely at such low temperatures. Large-size vitrification is associated with three competing needs on the CPA concentration. Since the cooling rate at the center of the specimen decreases with the increasing specimen size due to the scaling conductive resistance, higher CPA concentrations may be required to suppress crystallization in larger specimens. Higher CPA concentration generally requires lower cooling rates to avoid ice crystallization. On the other hand, since CPAs are potentially toxic, the lowest possible CPA concentration is required to maintain viability and facilitate functional recovery. The decrease in CPA concentration combined with an increase in cooling rates may intensify thermo-mechanical stress due to non-uniform thermal contraction to the point of structural destruction. Essentially, successful cryopreservation represents the outcome of an optimization problem on the composition and concentration of the CPA cocktail. The work presented in this thesis combines an experimental study on the thermal conductivity of relevant materials, and a theoretical study to identify the effects of the measured values on cryopreservation protocols. The unique contributions presented as the initial stage of the experimental study are: (i) the modification of the cryomacroscope and creation of an experimental program to make thermal conductivity measurements of CPA based on the existing transient hot wire technique, (ii) to develop a protocol for making thermal conductivity measurements during rewarming portion of the cryoprotocol, and (iii), to begin generating a data bank of thermal conductivity of CPA and materials used in cryopreservation. Thermal conductivity measurements are presented for the CPA Dimethyl Sulfoxide (DMSO), over a concentration range of 2M to 10M, in a temperature range of -180°C to 25°C. Samples of 2M to 6M DMSO were found to crystallize at quasi-steady cooling rates, while samples of 7.05 to 10M were found to vitrify. Thermal conductivities of the crystallized and vitrified material reach a tenfold difference at -180°C. The quality of measurements using the presented technique has been verified theoretically by means of finite element analysis (FEA) using the commercial code ANSYS. This experimental study is expanded to the study of thermal conductivity of the CPA cocktail DP6--a mixture of 3M DMSO and 3M propylene glycol, which has drawn significant attention in the cryobiology community in recent times. The unique contributions are the first thermal conductivity measurements reported in literature of the combined effect of DP6 with synthetic ice modulators (SIMs), including 6% 1,3Cyclohexanediol, 6% 2,3Butanediol, and 12% PEG400. Results of this study demonstrate that the thermal conductivity may vary by three fold between the amorphous and crystalline phases of DP6 below the glass transition temperature. Results of this study further demonstrate the ability of SIMs to decrease the extent of crystallization in DP6, even at subcritical cooling and rewarming rates. The accompanying theoretical investigation focuses on cryopreservation in a kidney model, in effort to explore how the thermal history is affected by variations in the measured thermal conductivity. This analysis is based on FEA using the commercial code ANSYS. In particular, the unique contributions of this study are: (i) thermal analysis of a vitrifying rabbit kidney based on an established rabbit-kidney cryopreservation protocol, and (ii), exploring scale-up thermal effects to a human-size organ. This represents a 21-fold increase in organ size. Results indicate that even in the case of the human kidney, cooling rates remain high enough in all parts of the kidney to prevent ice formation at temperatures above -100oC.

Orientador: Gisele Zoccal Mingoti / Banca: Gilson Hélio Toniollo / Banca: Ériklis Nogueira / Banca: Karina Beloti Avelino / Banca: Paulo Henrique Franceschini / Resumo: objetivo deste estudo foi contribuir para o conhecimento da técnica de criopreservação de embriões in vitro. Oócitos (n=965) foram maturados durante 24h em TCM-199 suplementado com 0,2mM de piruvato, 25 mM de bicarbonato de sódio e 75 mg/ml de gentamicina. Os zigotos foram cultivados em meio SOFm suplementado com 0,5% BSA e FCS 2,5%. Culturas foram realizadas a 38,5°C em 5% de CO2 no ar umidificado, em 100 μl de meio de gotículas recuperado com óleo mineral. No sétimo dia da cultura, os embriões (n = 387) foram marcados e apenas blastocistos excelente a boa qualidade foram criopreservados (10 embriões / palheta 0,25 ml). Os seguintes grupos experimentais foram concebidos, nomeado de acordo com a solução de crioprotetor utilizado: glicerol 1,0 M de etileno glicol em PBS (G); glicerol 1,0 M + 0,3 M de sacarose em PBS (GS); etilenoglicol 1.5 M em PBS (E) e 1,5 M + 0,3 M de sacarose em PBS (ES). Após o descongelamento, os embriões foram re-cultivadas em 100 ml de meio de gotas SOFm a 38,5 º C e 5% de CO2 no ar por 72 h. Os dados demonstraram que a qualidade do embrião, avaliada pelo escore de embriões e número de células embrionárias, parece ser melhor no grupo E e ES. No processo de vitrificação, 300 embriões de excelente qualidade morfológica, Bi e Bl foram vitrificados, e foram sincronizadas 500 receptoras de embriões, divididas em 3 grupos de 150 animais cada grupo. O diagnóstico de gestação nas receptoras após a inovulação dos embriões foram realizados após 42 dias. A taxa de concepção variou entre os grupos, sendo que no grupo I obteve 6 gestações (18,75%) de receptoras que receberam embriões da raça Nelore, 9 gestações (26,47%) de receptoras que receberam embriões da raça Brangus e 12 gestações (35,3%) de receptoras que receberam embriões da raça Brahman ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to contribute to the knowledge of the technique of cryopreservation of embryos in vitro. Oocytes (n = 965) were matured for 24h in TCM- 199 supplemented with 0.2 mM pyruvate, 25 mM sodium bicarbonate and 75 mg / ml of gentamicin. Zygotes were cultured in medium supplemented with 0.5% SOFm BSA and 2.5% FCS. Cultures were performed at 38.5 ° C in 5% CO2 in humidified air in 100 l of medium droplets recovered with mineral oil. On the seventh day of culture, the embryos (n = 387) were scored and only good quality excellent blastocysts were cryopreserved (10 embryos / straw 0.25 ml). The following experimental groups were designed, named according to the cryoprotectant solution used: glycerol 1.0 M ethylene glycol in PBS (G) 1.0 M glycerol + 0.3 M sucrose in PBS (GS), ethylene glycol 1.5 M PBS (E) and 1.5 M + 0.3 M sucrose in PBS (ES). After thawing, the embryos were re-grown in 100 ml of medium SOFm drops to 38.5 º C and 5% CO2 in air for 72 h. The data demonstrated that embryo quality was evaluated by scoring the number of embryos and embryonic cells, seems to be better in group E and ES. In the process of vitrification, 300 embryos of excellent morphology, Bi and Bl, were vitrified, and 500 were synchronized embryo recipients were divided into 3 groups of 150 animals each group. Pregnancies after embryo transfer in recipient embryos were performed after 42 days. The conception rate varied between the groups, whereas in group I had six pregnancies (18.75%) of recipients that received embryos Nellore, 9 pregnancies (26.47%) of recipients that received embryos of Brangus and 12 pregnancies (35.3%) of recipients that received embryos from Brahman ... (Complete abstract click electronic access below) / Doutor

This study investigated the development potential of equine embryos in vitro after biopsy and vitrification. Twenty embryos were obtained from Quarter Horse, Thoroughbred, and mix-breed light mares between three and ten years old. The twenty embryos were divided into a biopsy (n=10) and control group (n=10). The biopsy group underwent microaspiration biopsy using a micromanipulator to obtain a small tissue sample from the embryo. Both groups were then vitrified using a commercially available technique originally described by Carnevale (2006) at Colorado State \ University. All 20 embryos were cultured in DMEM/Hams F-12 medium under oil at 37°C in 5% CO2 in air (Hinrichs et al., 1990). Embryos were monitored for expansion and hatching. Embryo development was statistically different between the two groups (p<0.05). The biopsy procedure did result in a much lower development potential in the biopsy group as compared to the control group (20% vs. 80%). However, embryos in the biopsy group did show expansion and hatching therefore the combined procedure did not preclude development potential in vitro. Based on these findings, more research needs to be done to increase the success of the combined procedure and the ultimate viability of the embryos needs to be confirmed with the establishment of successful pregnancies.

For this research, the abnormality of tissue cultured plantlets,vitrification, was examined in Gypsophila paniculata.Measurement of the relative water content and water saturation deficit of plantlets in culture revealed that vitrified plantlets contain relatively more water and less air spaces than non-vitrified plantlets.The effect of relative humidity on vitrification and growth was investigated using a variety of methods.From the results found, it was determined the defining characteristic of vitrified plantlets is water filled intercellular spaces. It was also determined that the primary cause of vitrification is high relative humidity resulting in a lack of transpiration in vitro but that other factors such as unbalanced mineral nutrition or high medium cytokinin can exacerbate vitrification.Further research in tissue culture may investigate the influence of relative humidity on plant growth and morphology, the mechanism by which plants exclude water from their intercellular spaces and refine in vitro tissue mineral analysis as a means by which critical mineral concentrations can be determined. / Doctor of Philosophy (PhD)