Introduction
Vitrification as an embryo freezing method is successfully used for cryopreservation of embryos of different animal species as well as humans. For human embryos vitrification has been shown to be more favourable than slow freezing with regard to survival, ongoing pregnancy and implantation rates (Kuleshova & Lopata, Reference Kuleshova and Lopata2002; Stehlik et al., Reference Stehlik, Stehlik, Katayama, Kuwayama, Jambor, Brohammer and Kato2005). The survival rate after vitrification differs depending on the method of cryopreservation (Zhao et al., Reference Zhao, Quan, Zhou, Hou and Zhu2007), number of steps, cooling rates (Hredzák et al., Reference Hredzák, Ostró, Ždilová, Maraček and Kačmárik2006), type and concentration of cryoprotective agent (CPA) (Zhou et al., Reference Zhou, Zhou, Zhu, Hou, Jin, Zhao and Hong2007), developmental stage of embryo (Zhou et al., Reference Zhou, Hou, Jin, Yang, Yang, Quan, Tan and Zhu2005) as well as the embryo quality alone (Fabian et al., Reference Fabian, Gjorret, Berthelot, Martinat-Botté and Maddox-Hytel2005).
Several literature sources reported unfavourable ZP hardening as a possible reason of lower developmental potential of frozen/thawed embryos to hatching stage (Vanderzwalmen et al., Reference Vanderzwalmen, Bertin, Debauche, Standaert, Bollen, Van Roosendaal, Vandervorst, Schoysman and Zech2003). For this reason, different types of assisted hatching (AH) have been developed. AH can be performed by total mechanical and chemical penetration of ZP or partial enzymatic thinning of ZP by pronase enzyme or laser (Blake et al., Reference Blake, Forsberg, Johansson and Wikland2001). A study comparing these four different techniques showed similar implantation and pregnancy rates (Balaban et al., Reference Balaban, Urman, Alatas, Mercan, Mumcu and Isiklar2002).
Other non-invasive parameters for the testing of embryo viability seems to be the embryo diameter and total cell number (Mori et al., Reference Mori, Otoi and Suzuki2002; Makarevich et al., Reference Makarevich, Chrenek and Fl'ak2006). The measurement of embryo diameter and counting of cell number using nuclear staining with vital permanent dyes as Hoechst 33342 would enable the non-invasive selection of the embryos with best developmental potential without its destruction.
The present study aimed to compare directly: (1) the effectiveness of two vitrification procedures; and (2) the influence of mechanical and enzymatic assisted hatching techniques applied after embryo warming on the in vitro development, cell count and diameter of in vivo fertilized, in vitro cultured precompacted rabbit embryos.
Materials and Methods
Unless otherwise specified, chemicals were obtained from Sigma.
Recovery of rabbit embryos
Female New Zealand white rabbit does reared on the local farm were treated with PMSG (Werfaser, Alvetra and WERFF) i.m. at 20 IU/kg live weight, 72 h before mating. Immediately prior to mating, the females were injected i.m. with hCG (Werfachor, Alvetra und WERFFT) at 40 IU/kg live weight, afterwards the females were mated with a male of proven fertility from the same breed. The pronuclear stage eggs were flushed from the oviduct of slaughtered animals with phosphate-buffered saline (PBS, Gibco-Life Technologies) 19 to 20 h post coitum (hpc). The flushed eggs were evaluated morphologically and the eggs with both pronuclei were selected and cultured additionally for 24 h in vitro in a culture medium (k-DMEM, Gibco BRL) supplemented with 10% fetal calf serum (FCS, Gibco BRL.) in a humidified atmosphere of 5% CO2 at 39 °C.
Vitrification procedures
To avoid individual variations between does, the zygotes recovered from five does were used in five consecutive experiments. In vivo fertilized, in vitro cultured rabbit embryos at 8–12-cell stages were vitrified using one of the two procedures. In first procedure, a two-step cryoprotectant loading process was used. The vitrification solution (VS) composed of 40% ethylene glycol (EG) and 1 mol/l sucrose in the basic medium (HEPES-buffered CIM, Gibco BRL supplemented with 20% FCS). The precompacted embryos were then exposed to VS for 2 min and 30 s, respectively. In second procedure, an one-step loading process in the VS containing 40% EG, 18% Ficoll 70 and 0.3 mol/l sucrose (Papis et al., Reference Papis, Sypecka, Korwin-Kossakowski, Wenta-Muchalska and Bilska2005) dissolved in a basic medium was used. Vitrification solutions were equilibrated by prewarming at an ambient temperature. In both cases a group of embryos (maximum five) was put inside a French straw in a small volume of medium (0.5 μl) and directly plunged into liquid nitrogen. The straws were stored in liquid nitrogen for 1–3 days.
Warming process of vitrification and artificially assisted hatching (AH)
EG vitrified embryos were warmed using two-step dilution in sucrose solution. The embryos were expelled and plunged into 1 and 0.5 mol/l sucrose solution prepared in a basic medium, 5 and 5 min, respectively. EG + Ficoll vitrified embryos were directly expelled into 0.5 mol/l sucrose solution and equilibrated for 8 min. Immediately after warming, embryos were divided into two groups for either mechanical or enzymatic AH. For mechanical AH the zona pellucida of the embryo immobilized on a holding pipette was penetrated with a microinjection needle with a diameter of approximately 15 μm to make a single-holed zona puncture. Enzymatic AH was done by short (30 s) exposure of embryos to 0.5% (v/v) pronase solution in PBS. Developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida (hatching) after 3 days of in vitro culture in k-DMEM medium (Gibco BRL.) + 10% FCS in a humidified atmosphere of 5% CO2 at 39 °C.
Determination of cell count and diameter of rabbit embryos
Total cell number of blastocysts was determined using Hoechst 33342 staining and counting under a Leica fluorescence microscope, as was previously described (Chrenek et al., 2005). Embryo diameters, excepting ZP, were measured from the same images on the screen of the monitor using scale bar micrometer, which was previously calibrated on a ×40 objective and ×10 eye-pieces (Makarevich et al., Reference Makarevich, Chrenek and Fl'ak2006).
Statistical analysis
Chi-squared test was used to determine differences between two vitrification procedures as well as between the percentage of embryos developing to blastocysts and hatching blastocysts stage after mechanical or enzymatic AH. Data for embryo cell counts and diameters were expressed as mean values ± S.E.M. and assessed by analysis of variance (ANOVA) using Kruskal–Wallis test. Differences between groups at P < 0.05 were considered as significant.
Results
Effect of vitrification technique on the development, cell number and diameter of rabbit embryos
Table 1 summarizes survival and quality characteristics of rabbit embryos subjected to either of two procedures of vitrification at 8–12-cell stage. Blastocyst rate (22.5%) and hatching rate (15%) in the vitrified EG group were significantly lower than that for the vitrified EG + Ficoll group (63 and 63% resp.) and intact non-vitrified group (97 and 97% respectively). Total cell number was significantly lower (P < 0.05) in EG vitrified embryos compared to EG + Ficoll and the intact group. Embryo diameter was lower (P < 0.01) in EG vitrified group compared to the intact ones, whilst no difference was observed between EG + Ficoll vitrified and intact embryos.
Effect of vitrification method on the development, cell number and diameter (μm) of rabbit embryos
*P < 0.05 (chi-squared test, the values within the column differ significantly); **P < 0.05, (one–way ANOVA, Kruskal–Wallis test). Numbers in columns with different superscripts differ significantly.
Effect of assisted hatching (AH) on the development, cell number and diameter of rabbit embryos
Developmental rates for vitrified-warmed rabbit embryos following mechanical or enzymatic assisted hatching are compared (Table 2). In EG group with enzymatic AH and in both EG + Ficoll groups all blastocyst stage embryos (100%) reached hatching stage. However, in EG group with mechanical AH from 11 blastocysts only eight reached hatching stage. Total cell number in both vitrification groups (EG and EG + Ficoll), was lower (P < 0.01) when mechanical AH was applied compared to enzymatic AH. In embryo diameter significant (P < 0.05) differences were observed between mechanical and enzymatic AH using both vitrification procedures.
Effect of assisted hatching (AH) on the development, cell number and diameter (μm) of rabbit embryos
*P < 0.01 (chi-squared test), **P < 0.05 (one–way ANOVA, Kruskal–Wallis test). Numbers in columns with different superscripts differ significantly.
Discussion
Since the first cryopreservation study in rabbit, the best embryo survival rates have been obtained at the morula stage (Vicente et al., Reference Vicente, Viudes-De-Castro, Garcia and Baselga2003). This embryo stage allows the conventional freezing procedures to be substituted efficiently by a simple method of vitrification (Naik et al., Reference Naik, Rao, Vagdevi, Gnanprakash, Amarnath and Rao2005). Usually at in vivo experiments rabbit embryos after microinjection are cultured for further 24 h and then are transferred to recipients at the 8-cell stage. It is proper to use mucin coat-free embryos for micromanipulation only, which corresponds to 19–20 hpc. Following manipulations, mucin coat may be restored by transfer to the oviduct of synchronized recipients to increase their implantation potential. Transfer of in vitro manipulated and in vitro cultured embryos at higher stages than the blastocyst does not give satisfactory implantation rates. For this reason rabbit micro-manipulated embryos at 8-cell stage are more proper for the cryopreservation and embryo bank establishment. Precompacted (1- to 8-cell stage) embryos are more sensitive to external influences such as cryopreservation than post-compacted and differentiated embryos. This stage-dependent viability of vitrified rabbit embryos was reported in an earlier report (Smorag et al., Reference Smorag, Gajda, Wieczorek and Jura1989). For this reason a choice of effective vitrification procedure with high post-thaw survival rates is very important for the embryo bank foundation.
Important factors affecting success of vitrification include rates of temperature decrease and cooling of vitrification solutions. Hredzák et al. (Reference Hredzák, Ostró, Ždilová, Maraček and Kačmárik2006) observed a decrease in hatching rate of blastocysts produced from 8-cell mouse embryos vitrified in straws (8%) compared to the group frozen in pipetting tips (38%) or introduced dropwise directly into liquid nitrogen (60%). An important role of cooling rates of the solution is ascribed to the freezing container, thermal conductivity of its walls and the volume of vitrification solution (Liebermann et al., Reference Liebermann, Dietl and Vanderzwalmen2003). We used, as a carrier system, classical French straw inside which we put the group of embryos at a minimum volume (<1.0 μl) to meet this requirement. The embryos adhered to the straw wall during the cooling process and were expelled to the thawing medium without drop-outs.
Penetrating cryoprotectants such as propylene glycol or ethylene glycol that require the highest concentrations to vitrify and displace the most water are actually the least toxic. In our study the vitrification solution based on ethylene glycol at concentration of 40% (v/v) in basic medium had inferior effect on post-thaw viability of rabbit precompacted embryos. The addition of macromolecular substance Ficoll (mol. wt 70,000) to vitrification medium improved the survival and blastocyst rate of vitrified/warmed embryos to 67% in comparison with 22.5% in the EG group. This polymer is less toxic and can protect embryos against cryoinjury by mitigating the mechanical stress occurring during cryopreservation, and by building a viscous matrix around these embryos, which prevents crystallization during cooling and warming (Kuleshova et al., Reference Kuleshova, Shaw and Trounson2001). Vicente & Garcia-Ximénez (Reference Vicente and Garcia-Ximénez1994) observed worst survival rate of the cryopreserved rabbit morulae and development to blastocysts in vitro in the EG-treated embryos compared to mixture of ethylene glycol and dimethylsulfoxide (EG + DMSO, 20% v/v).
Hardening of ZP may occur naturally following fertilization in order to block polyspermic fertilization, after prolonged exposure of embryos to artificial culture conditions and after cryopreservation (De Vos & Van Steirteghem, Reference De Vos and Van Steirteghem2000). Popelková et al. (Reference Popelková, Chrenek, Pivko, Makarevich, Kubovičová and Kačmárik2005) observed high significant differences in hatching rates between OPS vitrified rabbit embryos (56%) and non-frozen ones (94%). This decrease in the hatching rate may reflect altered viability of the thawed embryos or structural changes of the zona. Studies specifically addressed the issue of AH performed on frozen–thawed embryos reported controversial results. Kong et al. (Reference Kong, Lee, Im, Cho, Ohh, Ohh and Bae2000) declared that enzyme-assisted hatching following vitrification enhances post-thaw hatching rates of bovine in vitro produced embryos. Ng et al. (Reference Ng, Naveed, Lau, Yeung, Chan, Tang and Ho2005) compared implantation and pregnancy rates of frozen–thawed embryo transfer (FET) cycles with and without laser AH and concluded that laser AH does not improve the implantation rate of FET cycles. Balaban et al. (Reference Balaban, Urman, Alatas, Mercan, Mumcu and Isiklar2002) found similar implantation and pregnancy rates for four different techniques of AH including acidic Tyrode's solution, partial zona dissection, diode laser and pronase zona thinning. In the present study, the beneficial effect of total mechanical ZP breaching as well as enzymatic ZP thinning AH after vitrification was not evident as no significant increase in the blastocyst hatching rate during post–warm in vitro culture was recorded. The study of Mantoudis et al. (2001) and Blake et al. (Reference Blake, Forsberg, Johansson and Wikland2001) showed a higher hatching rate when AH was performed by zona thinning rather than by complete zona breaching. These studies suggest that the opening the zona pellucida may have adverse effects, such as abnormal blastocyst expansion, loss of blastomeres through the breached zona and lack of the protective effect of the ZP against infectious agents. In our in vitro study both techniques of AH had no adverse effect on the further development of rabbit embryos in culture.
Makarevich et al. (Reference Makarevich, Chrenek and Fl'ak2006) tested the effect of microinjection (Mi) of foreign gene into rabbit egg pronucleus and epidermal growth factor (EGF) addition on the blastocyst rate, total cell number and the diameter of embryos. The microinjection procedure in their study decreased blastocyst rate and cell number, but did not affect the embryo diameter. In our study the embryos vitrified by EG had lower total cell number and embryo diameter compared to intact non-vitrified ones, and also showed lower total cell number compared with EG + Ficoll group. The EG vitrified group showed also significantly lower blastocyst and hatching rate. Most of embryos did not develop further after thawing.
In conclusion, the one-step vitrification procedure with EG and Ficoll used as cryoprotective agents gave superior results of post-thaw developmental potential of precompacted stage rabbit embryos cultured in vitro. The two-step vitrification with EG based solution showed to be less effective in terms of hatching rate and quality of embryos (cell number and embryo diameter). No influence of either mechanical or enzyme-assisted hatching on the post-thaw viability and quality of rabbit embryos in vitro was observed.
Acknowledgement
This work was supported by the Slovak Research and Development Agency under the contract no. LPP-0126–06 and by the grant from the project of the Ministry of Education, Slovak Republic, no. 1985/2007.
