This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbecco's phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v−1) of CRYO3 or 18% (v.v−1) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0.066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions.
Citation: Bruyère P, Baudot A, Joly T, Commin L, Pillet E, Guérin P, et al. (2013) A Chemically Defined Medium for Rabbit Embryo Cryopreservation. PLoS ONE 8(8): e71547. https://doi.org/10.1371/journal.pone.0071547
Editor: Berta Alsina, Universitat Pompeu Fabra, Spain
Received: February 15, 2012; Accepted: July 1, 2013; Published: August 20, 2013
Copyright: © 2013 Bruyère et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Embryo transport and transfer in domestic animals is widely used because it is considered to be safest, less expensive and more respectful to animal welfare than animal transport . In 2010, more than 930,000 in vivo- or in vitro-produced bovine embryos were transferred worldwide, including more than 350,000 frozen/thawed embryos .
However, although embryo transfer is considered to be safe according to the recommendations of the International Embryo Transfer Society, sanitary risks still exist , , . One of these concerns is the source of macromolecules added to the various media used for embryo transfer , including the cryopreservation solutions. In fact, the most frequently used sources for cryopreservation are animal-derived products, such as foetal calf serum (FCS) or bovine serum albumin (BSA), which may be contaminated by pathogens, particularly by viruses , , . Among these viruses, Bovine Viral Diarrhoea virus is the most important cause of concern because of its large economic impact , its prevalence in FCS , its relative resistance to gamma irradiation  and cryopreservation procedures , and consequently the associated risk of disease transmission to recipients by an infected embryo . Prion diseases can also probably be transmitted through blood-derived products –, especially as it is difficult to inactivate the transmissible degenerative encephalopathy agents with the usual processing of animal-based products . In addition to the sanitary risks, undefined compounds, especially peptides, may be bound to albumin and may induce variability in the composition of the FCS or BSA, causing deleterious effects on embryos .
Although using BSA lowers the sanitary risks , , the safest practice is to use substitutes that are not biologically derived , . Sodium hyaluronate ,  and vegetable peptones  have been successfully used as substitutes for animal products in embryo cryopreservation solutions. Acceptable but less consistent results have also been obtained with polyvinyl alcohol –. The use of polyvinylpyrrolidone ,  or surfactants (VF 5 , Pluronic F68 ) has led to less conclusive results. Dextran  and ficoll ,  have also been added to embryo cryopreservation solutions without any animal-derived macromolecular components to reduce the concentrations of cryoprotectants.
However, the use of substitutes may lead to variations in the physical properties of the cryopreservation solutions that could be deleterious for the embryos. Consequently, these variations need to be controlled. Differential Scanning Calorimetry (DSC) is an interesting physical analysis tool that has been used in cryobiology to characterise cryopreservation solutions , , to optimise cryopreservation protocols –, and to better understand ice formation in cells, embryos and other living organisms –. In fact, DSC allows the dynamic evaluation of thermal properties of cryopreservation solutions, particularly during crystallisation and melting. For slow-freezing solutions, the thermodynamic characteristics, such as the phase transition temperature and the quantity of ice crystallised and melted, can thus be measured.
In a preliminary study, Joly obtained promising results in rabbit (Oryctolagus cuniculus) embryo cryopreservation with a chemically defined substitute that contained no animal products (CRYO3, Ref. 5617, Stem Alpha, France) . The aim of this study is to more thoroughly evaluate this potential substitute with two consecutive approaches. First, DSC was used to compare selected thermodynamic characteristics of cryopreservation solutions containing the potential substitute (CRYO3) with those of standard cryopreservation solutions containing FCS. Second, the substitute was evaluated in a biological setting by quantifying the viability after (i) in vitro culture and (ii) embryo transfer of rabbit embryos which were previously frozen in a solution containing the substitute or a FCS-based solution.
Thermodynamic characteristics of the cryopreservation solutions containing FCS.
As shown in Table 1, the mean values of the end of melting temperature (Tm) and the enthalpy of crystallisation (ΔH)