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Introduction
Rabbit zygotes and early embryos have been successfully developed to blastocysts using different culture media (Saenz-de-Juano et al., 2011). However, these media do not mimic the oviductal environment and in vitro developed embryos differ from their in vivo analogues. Specifically, in vitro cultured rabbit embryos show fewer cells, smaller diameters (Adams, 1970) and morphological signs of degeneration after one day in culture from morulae to early blastocysts (Hegele-Hartung et al., 1988). Thus, embryos undergo a retarded development under in vitro conditions (Carney and Foote, 1990).
The female rabbit has certain physiological and anatomical characteristics that make it especially suitable for the application of embryo reproductive techniques (García, 2018). In vitro embryo culture is an assisted reproductive technique used frequently in embryo biology. The comparative features of embryos that develop under in vitro and in vivo conditions are particularly important for designing embryo transfer procedures that fulfil embryo-recipient synchronization requirements. Therefore, in order to maximize the results of the application of reproductive techniques, it is necessary to accurately determine the degree of delay in embryonic development of cultured embryos. Therefore, the aim of this study was to determine the degree of asynchrony in embryo development between in vitro and in vivo embryos at 72 hpc in rabbits.
Materials and Methods
Ethical considerations
All experimental procedures were approved by the Committee of Ethics and Animal Welfare of the Miguel Hernández University, Spain (Ref: 2019/VSC/PEA/0017).
In vivo embryo production and collection
Non-lactating multiparous female rabbits were used (Argente et al., 2019). A total of 41 and 14 does were mated and then slaughtered at 48 and 72 hours post-coitum (hpc) by intravenous administration of 50 mg sodium thiopental/kg body weight (thiobarbital, B. Braun Medical S.A., Barcelona, Spain). The ovaries, oviducts and uterine horns were removed. To recover the embryos, the oviduct and the first third of the uterine horn were flushed with 5 mL Dulbecco phosphate buffered saline (DPBS, Sigma, Alcobendas, Madrid, Spain) supplemented with 0.2% (wt/vol) bovine serum albumin (BSA, Cod. A-3111, Sigma) and 0.2 mL of antibiotic (Penivet 1, Divasa Farmavic, Barcelona, Spain) at room temperature. In vivo embryos were examined. They were considered normal when homogenous cellular mass and spherical zona pellucida and mucin coat was present. A binocular stereoscopy microscope (Leica Mz 9.5-600x, Wetzlar, Germany) was used. At 48 hpc, normal embryos were classified as 16-cells or early morulae (Figure 1A). At 72 hpc, normal embryos were classified as early morulae or compacted morulae (Figure 1B).
Embryo culture
Normal embryos at 48 hpc from each doe were cultured in a one-well 4-well embryo culture dish (NUNC A/S, Thermo Fischer Scientific, Denmark) containing 1 ml culture media (TCM- 199 supplemented with 10% fetal bovine serum). Culture was performed at 38.5 ºC in 5% CO2 in air saturated humidity. In vitro developed embryos were examined after 30 or 32 h of culture. So, the asynchrony between in vivo and in vitro embryos was +6 h or +8 h. Then, embryos were classified as early morulae or compacted morulae. A total of 20 and 21 females were used to culture their embryos with an asynchrony of + 6h and + 8h, respectively.
Statistical analyses
Early morulae and compacted morulae were expressed as a percentage of normal embryos. Differences in asynchrony between in vivo and in vitro embryos were estimated with a model including the effects of asynchrony (in vivo embryos, and in vitro embryos with +6 h and +8 h of asynchrony). Analysis was performed using Bayesian methodology. Bounded uniform priors were used for all effects. Residuals were, a priori, normally distributed with mean 0 and variance Iσ2e. The prior for the variance was also bounded uniform. Features of the marginal posterior distributions for all unknowns were estimated using Gibbs sampling. Inferences were derived from the marginal posterior distributions. Median, difference between in vivo and in vitro embryos (D), and the shortest interval with 95% probability of containing the true value (HPD95%) were provided. The HPD95% showed the precision of the estimation and can be asymmetric around the estimation. The actual probability of D higher than zero (P) was estimated. The Rabbit program developed by the Institute for Animal Science and Technology (Valencia, Spain) was used.
Results
Table 1 shows differences between in vivo and in vitro embryo development. When the asynchrony was +6 h, the percentage of early morulae was lower in in vivo than in in vitro embryos (-40.7%, P = 1). So, the percentage of compacted morulae was higher. An asynchrony of +6 h was not enough for matching in vitro and in vivo embryo development. Nevertheless, the percentage of early morulae (-7.0%, P = 0.68) and compacted morulae were similar between in vivo and in vitro embryos with +8 h of asynchrony.
Figure 1 A) Scaled in vivo early morulae obtained at 48 hpc. B) Scaled compacted morulae from in vivo embryos at 48 hpc and cultured for 32 h.
Table 1 Differences between development between in vivo and in vitro embryos.
| In vivo | In vitro | D | HPD95% | P | |
|---|---|---|---|---|---|
| +6 h asynchrony (N) | 14 | 20 | |||
| Early morulae | 32.8 | 73.5 | -40.7 | -65.0, -16.3 | 1.00 |
| Compacted morulae | 67.2 | 26.5 | 40.7 | 16.1, 66.2 | 1.00 |
| + 8 h asynchrony (N) | 14 | 21 | |||
| Early morulae | 32.8 | 39.8 | -7.0 | -36.0, 18.3 | 0.68 |
| Compacted morulae | 67.2 | 60.2 | 7.0 | -20.1, 34.1 | 0.68 |
N=number of does; In vivo=median of in vivo embryos at 72 hpc; In vitro=median of in vitro embryos; D=median of the difference between the in vivo and in vitro embryos; HPD95%=highest posterior density region at 95%; P=probability of the difference being ˃0 when D˃0 and probability of the difference being <0 when D<0.
Discussion
The low development of cultured embryos also occurs in cattle (Lonergan et al., 2016) and pigs (Fowler et al., 2018). While embryo culture systems are static, in vivo embryos are exposed to a constantly changing environment as it passes along the oviduct to the uterus. Concomitantly, embryos exhibit changes in physiology and energy metabolism between fertilization and blastocyst (Gardner, 1998). Thus, embryo manipulation and adaptation to in vitro conditions as its requirements change during development lead to a reduction in development (García, 2018). We have determined that asynchrony in rabbits is 8 hours between cultured and in vivo embryos. This result can be used to optimize the design of experiments in which cultured embryos are used.
In conclusion, development was lower in in vitro than in in vivo embryos. Asynchrony between in vivo and in vitro embryonic development was 8 hours.
