New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species

International audienc

The Use of Chitooligosaccharides in Cryopreservation: Discussion of Concept and First Answers from DSC Thermal Analysis

The use of dimethyl sulfoxide (Me2SO) as a cryoprotectant agent (CPA) is controversial. Indeed, this cryoprotectant agent (CPA) is cytotoxic and potentially mutagenic. Therefore, other cryoprotectants must be used to reduce the proportion of Me2SO in slow-freezing solutions. In this chapter, we propose to present the first evaluation of new non-penetrating cryoprotectants: the chitooligosaccharides (COS). These molecules are chitosan oligomers, which are biocompatible, antioxidant, and bacteriostatic. We first review the use of saccharides through cryopreservation processes. We question the possibility to reduce penetrating CPA during slow-freezing procedures. We propose to use COS as extracellular CPA to reduce the use of Me2SO. We question the biocompatibility of COS on mouse embryos through the analysis of the cells’ development. Next, we evaluate these molecules in slow-freezing solutions with a reduced quantity of Me2SO. Our experimental approach is a physical method often used to characterize slow-freezing solutions. Differential scanning calorimetry (DSC) allows to evaluate the crystallization and melting processes, the amount of crystallized water, and the equilibrium temperature and consequently to evaluate the impact of different cryoprotectants. This study gives a better understanding on how slow-freezing protocols could be improved with extracellular CPA

Cryopreservation of ovarian tissue and embryo production in the bitch

De nos jours, la cryoconservation est une technique très largement utilisée dans les protocoles d’assistance à la reproduction ou comme outil pour la sauvegarde des ressources génétiques. Toutefois, la chienne est un modèle animal complexe pour l’application des biotechnologies de la reproduction du fait de ses nombreuses singularités anatomiques et physiologiques. L’objectif de notre travail était d’étudier et de développer une méthode de cryoconservation des ressources génétiques chez la chienne par le biais de deux types de ressources : les embryons et le tissu ovarien. Après avoir mis au point une méthode de collecte d’embryons, nous nous sommes appliqués à la constitution d’un stock d’embryons cryoconservés en prévision d’un transfert embryonnaire. L’étude et le développement d’un protocole de cryoconservation du tissu ovarien ont été abordés après avoir adapté et validé nos méthodes d’analyses in vitro. L’utilisation de plans d’expériences factoriels fractionnaires a permis de mettre en évidence les facteurs les plus influents sur la qualité de la réserve folliculaire (nature du cryoprotecteur pénétrant, cinétique de congélation, étapes d’équilibration) et de proposer un protocole de cryoconservation. La combinaison du DMSO incorporé en un seul bain d’équilibration avec une vitesse de congélation de 0,3°C/min est apparue comme la combinaison la plus appropriée à la cryoconservation de tissu ovarien chez la chienne et a permis d’observer, après xénogreffe de tissu ovarien cryoconservé, une reprise de la croissance folliculaire et de l’activité hormonale du tissu grefféNowadays, cryopreservation is widely used in animal assisted reproduction or safeguarding of genetic resources. Nevertheless, the bitch is a complex animal model concerning the use of this biotechnology, due to numerous anatomical and physiological peculiarities. The aim of our research work was to investigate and develop a method of cryopreservation of genetic resources in the bitch by exploring two kinds of resources: embryos and ovarian tissue. After the setting up of a method for embryo collection, we have built up a stock of cryopreserved embryo for subsequent embryo transfer. After a preliminary validation of our in vitro assessment methods, the investigation and development of a cryopreservation protocol has been conducted. The use of fractional experimental design allowed us to highlight the main factors affecting the follicular pool quality (CPA nature, freezing rate and equilibration steps). The combination of DMSO incorporated in a unique equilibration bath with a freezing rate of 0.3°C/min appeared to be suitable for the cryopreservation of bitch ovarian tissue. Finally, Follicular growth and hormonal activity resumption have been observed after xenotransplantation of cryopreserved bitch ovarian tissu

Comparison Between an Animal-Derived Product Medium and a Chemically Defined Medium for Ram Sperm Cryopreservation

International audienceAnimal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, tend to be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617; Stem Alpha, Saint-Genis-l'Argentiere, France), 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 effect of a CRYO3-based medium on ram sperm freezing regarding in vitro parameters and in vivo fertility. Semen from nine Charolais rams was collected using an artificial vagina, then split and frozen using two media: a CRYO3-based medium or a control medium containing egg yolk (10%) and milk (45%). Sperm membrane integrity (propidium iodide [PI]/SYBR-14 and calcein AM/ethidium homodimer-1), acrosome integrity (FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, while functional membrane integrity was assessed using a hypo-osmotic swelling test and motility parameters, evaluated by computer-assisted sperm analysis. Pregnancy rates, prolificacy, and the average daily weight gain (DWG) of lambs were evaluated after performing 195 laparoscopic inseminations. The control medium showed significantly higher results than CRYO-based medium for all in vitro parameters, except for linearity and straightness (motions parameters). Conversely, field trials showed no significant difference between the control medium and the CRYO3-based medium for pregnancy rates (72.2% and 67.9%, respectively), prolificacy (1.8 and 1.6, respectively), and the DWG (0.34 and 0.35 kg/d, respectively). This preliminary study showed that CRYO3 cannot replace egg yolk and milk in freezing extenders for commercial purposes. However, as laparoscopic inseminations allowed a 67% pregnancy rate, CRYO3-based medium remains an option for international transport or long-term storage of genetic diversity

Comparative Evaluation of the In Vitro Cytotoxicity of a Series of Chitosans and Chitooligosaccharides Water-Soluble at Physiological pH

International audienceChitosans (CS) have been of great interest due to their properties and numerous applications. However, CS have poor solubility in neutral and basic media, which limits their use in these conditions. In contrast, chitooligosaccharides (COS) have better solubility in water and lower viscosity in aqueous solutions whilst maintaining interesting biological properties. CS and COS, unlike other sugars, are not single polymers with a defined structure but are groups of molecules with modifiable structural parameters, allowing the adaptation and optimization of their properties. The great versatility of CS and COS makes these molecules very attractive for different applications, such as cryopreservation. Here, we investigated the effect of the degree of polymerization (DP), degree of N-acetylation (DA) and concentration of a series of synthesized CS and COS, water-soluble at physiological pH, on their cytotoxicity in an L929 fibroblast cell culture. Our results demonstrated that CS and COS showed no sign of toxicity regarding cell viability at low concentrations (≤10 mg/mL), independently of their DP and DA, whereas a compromising effect on cell viability was observed at a high concentration (100 mg/mL)

Relevant facts about SnoMax when used in DSC experiments

International audienc

Ice induction in DSC experiments with Snomax®

International audienceFor differential scanning calorimetry (DSC) analysis, controlling the degree of supercooling at which crystallization begins may be required for several studies, experiments, and applications. In this paper, the use of Snomax, an ice nucleating agent (INA), was evaluated to create ice at a desirable temperature range in a DSC aluminum sample pan. The effect of Snomax on the nucleation temperature (Tn) was studied in pure water. Best practices and methods are described in terms of the Tn dependence on three experimental parameters: (i) the Snomax concentration that controls the Tn value for different groups according to the three classes of the Pseudomonas syringae protein aggregates (from which Snomax originates); (ii) the sample volume that affects the presence probabilities of the different INA subpopulations in the solution and that could also favor their deterioration; and (iii) the cooling rate that does not seem to further affect the Tn value. There is no-evidence of time dependence of the nucleation process promoted by Snomax. The presence of artifacts or disturbances introduced by the addition of Snomax into the solution was evaluated. No major disturbances of the thermodynamic characteristics of these solutions were observed with the addition of Snomax below 103 mg L−1 concentration. This underscores the possible use of Snomax for controlling ice nucleation during DSC experiments

L'insémination sous laparoscopie permet chez la brebie d'utiliser un milieu de congélation dénué de produits d'origine animale

International audienceAnimal-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