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Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0&ndash, 5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.

International audience; Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, must be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617, Stem Alpha), called CRYO3, is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effects of a CRYO3-based medium and of two cooling rates on in vitro parameters and in vivo fertility of ram sperm. Six rams (Blanche du Massif Central) were subjected to sperm collection four times using an artificial vagina. Sperm were split and frozen in three media: an egg yolk and milk-based medium (positive control), a CRYO3-based medium (tested medium), and a medium without additives (negative control). The two cooling rates were related to the distance between the straws and the surface of liquid nitrogen during the freezing process (5 and 20 cm). Sperm membrane integrity (propidium iodide/SYBR-14), acrosome integrity (fluorescein isothiocyanate-peanut agglutinin/propidium iodide; FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, whereas functional membrane integrity was assessed using a hypo-osmotic swelling test and motion characteristics were evaluated using computer-assisted sperm analysis. Pregnancy rate, parturition rate, and prolificacy were evaluated after performing laparoscopic inseminations (n ¼ 75 ewes). Moreover, we characterised the freezing media thermodynamically using a differential scanning calorimeter. Statistical analyses were performed using R software. In vitro parameters were assessed using a mixed model including the time and the medium as fixed effects and the ram as a random effect. Pregnancy and parturition rates, following a binomial distribution, and prolificacy, assumed to follow a Poisson distribution, were analysed using generalised linear models, including the medium as a fixed effect and the ram as a random effect. Differences with P , 0.05 were considered statistically significant. The cooling rates had no significant effect except on the wobble motion parameter. The positive control medium showed significantly higher results than the CRYO3-based medium and the negative control medium for all in vitro parameters except for straightness motion parameter. Conversely, field trials showed no significant difference between the media for pregnancy rate (71, 64, and 74%), parturition rate (68, 61, and 74%) and prolificacy (2.0, 2.1, and 1.7), for the positive control, CRYO3-based medium, and the negative control, respectively. This study showed that the product, CRYO3, cannot replace egg yolk and milk in freezing extenders. Moreover, we showed that laparoscopic inseminations allowed a 74% parturition rate due to an easy and inexpensive medium comprising only a Tris buffer and glycerol. Although it could not be used on a large scale, this medium remains an option for international transport or long-term storage of genetic diversity.

Long-term antibiotic-free preservation of rainbow trout milt (oncorhynchus mykiss). 7. International Workshop on the Biology of Fish Gametes

Changes in sperm cryopreservation procedure for industrial needs. 7. International Workshop on the Biology of Fish Gametes

Session : Reproduction; Cryoconservation du sperme de salmonidés : évolution des pratiques et amélioration des fécondances. 6. Journées de la Recherche Filière Piscicole

Nowadays, reproductive (embryos, sperm, oocytes) and somatic (fibroblasts and pluripotent stem cells) resources are cryopreserved in media containing animal-derived products (serum, egg yolk, milk). Using these products raises sanitary (risk of contamination) as well as scientific concerns (reproducibility limits due to the variability of their composition). This study aims to replace animal derived-product in assessing the effect of a synthetic and chemically defined medium, STEMALPHA.CRY03® (Stem Alpha, France), on the cryopreservation of ovine and bovine sperm, and on rabbit pluripotent stem cells. First, a physical approach permitted to study the cooling rates and the characterization of thermodynamic properties of the freezing media. The differential scanning calorimetry allowed us to define their phase transition temperatures (crystallization temperature, melting temperature and enthalpy variation of crystallization, proportional to the amount of crystallized ice). Second, a biological approach was used for the cryopreservation of bovine and ovine sperm, as well as rabbit pluripotent stem cells. Flow cytometry and computer- assisted sperm analyses showed that STEMALPHA.CRY03® impaired bovine sperm, compared to a medium containing animal derived-product. These last results were confirmed in ovine species. Nevertheless, artificial insemination by laparoscopy (n = 270 ewes) counteracts this impairment and allowed an average pregnancy rate of 70 %. Moreover, without any additive in the freezing medium, a similar pregnancy rate was obtained. The study of pluripotent gene expression profile, and analyses of viability and growth rates for the cryopreservation of rabbit pluripotent stem cells confirmed that synthetic media, STEMALPHA.CRY03® (with 4, 5 or 10 % of cryoprotectant) and CryoStor® CS10 (containing 10 % of cryoprotectant) were more efficient than serum-based media. We demonstrate that it is possible to cryopreserve sperm cells and pluripotent stem cells in synthetic and chemically defined media. 0ur results confirmed the interest of a standardized approach for cryopreservation procedures of genetic resources in mammals. This work meets the needs of cryobanking activities (quality policy) and of the regulation development within the framework of international trade, Cette thèse s’inscrit dans le cadre du projet CRB-Anim (Centre de Ressources Biologiques d’Animaux Domestiques) dont le but est de constituer une cryobanque nationale et d’améliorer les techniques de cryoconservation. Aujourd’hui, les ressources biologiques de type reproductif (embryons, sperme, ovocytes) et de type somatique (fibroblastes et cellules souches pluripotentes) sont conservées dans des milieux contenant des produits d’origine animale (POA). L’utilisation actuelle des POA (sérums, lait, jaune d’œuf) pose des problématiques sanitaires (risque de contamination) et scientifiques (manque de reproductibilité liée à la variabilité de composition des POA). Cette étude a pour objectif d’évaluer l’effet d’un milieu de préservation synthétique, chimiquement défini, breveté pour la congélation de cellules de sang de cordon (STEMALPHA.CRYO3) sur la cryoconservation de sperme ovin et bovin, et de cellules souches pluripotentes de lapin. Pour cela, une approche biologique (étude in vitro et in vivo sur le terrain), alliée à une approche physique (étude des cinétiques de refroidissement et caractérisation des propriétés thermodynamiques des milieux de congélation) ont été mis en place.Nos résultats montrent l’intérêt de l’utilisation du STEMALPHA.CRYO3, en tant que substituant aux sérums, dans les solutions de cryoconservation des cellules souches pluripotentes. Néanmoins, notre étude indique que ce produit n’est pas efficace pour protéger le sperme lors de la congélation. Cette thèse confirme l’intérêt de standardiser les procédures de cryoconservation afin d’assurer la qualité des ressources biologiques pour les cryobanques et les échanges internationaux