Insights into Species Preservation: Cryobanking of Rabbit Somatic and Pluripotent Stem Cells.

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.

Rapid cooling of rabbit embryos in a synthetic medium.

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.

INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling.

Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes produced from EY phospholipids on stallion sperm storage at 4°C. The sperm of stallions from two studs was diluted in INRA96 extender (as control) or an experimental extender (EE) composed of INRA96 supplemented with liposomes of EY phospholipids. After 24H (D1), 72H (D3), and 6 days (D6) or 7 days (D7), motility parameters were evaluated using Computer Assisted Semen Analyzer. Our results demonstrated that total and progressive motility decreased significantly after dilution and storage in INRA96 between D1 and D3 (P < .05) while no significant decrease was observed between D1 and D3 with EE. Regarding VAP parameter, no significant difference was observed between extenders except at D7 in stud 2. Moreover, total and progressive motility were maintained at a significantly higher level (D3, D6, D7) when sperm was stored in EE compared to INRA96. These promising results demonstrate that the supplementation of INRA96 extender with egg-yolk phospholipids liposomes allows a higher protection to stallion sperm cells.

The effect of adjusting settings within a Computer-Assisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results.

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.

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

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.