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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 (0-5%) 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.

Animal-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'Argentière, 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.

Embryo cryopreservation media usually contain animal-derived products, such as bovine serum albumin (BSA). These products present two major disadvantages: an undefined variable composition and a risk of pathogen transmission. We aimed to evaluate the effect of replacing BSA in rabbit embryo rapid cooling "freezing" and warming media with a chemically defined medium with no animal-derived products: STEM ALPHA. Cryo3 ("Cryo3"). A total of 1540 rabbit morulae were divided into three cryopreservation groups (group 1: BSA, group 2: 20% Cryo3 and group 3: 100% Cryo3) and a fresh controls group. After rapid cooling, embryos were cultured (in vitro approach), or transferred into synchronized does (in vivo approach). In the in vitro approach, post-warm survival rates obtained with 100% Cryo3 (94.9%) were superior to BSA (90.8%) and 20% Cryo3 (85.6%). The blastocyst formation rate was similar between BSA, 20% Cryo3 and 100% Cryo3 groups (85.1, 77.9 and 83.3%, respectively), as was the expansion/hatching rate (63.1, 63.4 and 58.0%, respectively) and embryo mitochondrial activity. In the in vivo approach, pregnancy (80.0, 68.0 and 95.2%, respectively), implantation (40.5, 45.9 and 44.8%, respectively), and live-foetus rates (35.6, 35.5 and 38.1%, respectively) were similar between the three groups. To conclude, Cryo3 can replace BSA in rabbit embryo rapid cooling "freezing" and warming media., (Copyright © 2018 Elsevier Inc. All rights reserved.)