In vitro and in vivodevelopment of mice morulae after storage in non-frozen conditions
© Hourcade et al.; licensee BioMed Central Ltd. 2012
Received: 8 March 2012
Accepted: 7 August 2012
Published: 22 August 2012
Interchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment.
Mouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR.
In vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further characterize the effect of non-frozen storage combining a molecular approach with the ordinary in vitro culture evaluation, embryos collected at the morula stage were submitted to the same storage conditions described throughout 48 hours. In vitro culture of those embryos showed a significant decrease in their developmental rate to blastocyst in both KSOMeq and KSOM-H at 15degrees C, which also affected the total number of cells. Gene transcription studies confirmed significant alterations in retrotransposons (Erv4 and Iap) after 48 h of storage at 15degrees C.
Our results show that both KSOMeq and KSOM-H can be equally used, and that several temperature conditions allow good survival rates in vitro and in vivo. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24–48 hours, providing a reliable and less demanding technical alternative for embryo interchanges.
KeywordsEmbryo Mouse Storage
An increasing number of genetically modified (GM) mice are exchanged between laboratories every year. National and international legislations enforce reducing the number of laboratory animals used for research purposes, and one clear way to accomplish this goal is to avoid the duplication of a GM line that has been already generated. Unfortunately, many of these lines are not stored in international repositories where cryobanking is well-established, but they are kept alive in animal facilities. Transport of live animals poses certain risks: not only may it compromise the health status of the receiving facility, but there is also an animal welfare issue and increasingly restrictive conditions from air and ground carriers.
On the other hand, cryopreserved embryos allow safe transport, but this approach demands a certain degree of expertise to freeze and thaw them correctly . Moreover, the containers used are costly and should be returned with the concomitant expense. Some approaches have explored the feasibility of alternative methods using, for instance, intermediate recipients [2–4], or specific temperatures [5, 6]. In these studies, 2-cell embryos were used, even though embryos of certain mice strains are prone to 2-cell blockage when cultured in suboptimal conditions . A simple way to overcome this risk is to use embryos in later stages of development, like morulae, that still allow 48 hours of autonomy. This time span is enough to reach most destinations and can easily complement cryopreservation without a need for specific equipment and expertise in cryobiology. Transport of embryos in liquid stage can be achieved either in culture media or in standard embryo holding media like M-2, where bicarbonate is substituted with HEPES. With this replacement pH is stabilized when embryos are handled; however, bicarbonate is necessary for the optimal development of embryos and long-term exposure to holding media might be detrimental.
Our aim was to first explore different storage conditions, trying to simplify the technical skills needed in order to promote the use of embryos for exchanging GM lines, even with facilities unfamiliar with thawing procedures. Secondly, we tried to characterize how gene expression patterns of some relevant genes during preimplantational development are affected by storage conditions.
Reagents and Media
All chemicals and media were purchased from Sigma Chemical Co. (Madrid, Spain), unless otherwise stated.
Female B6CBAF1 mice aged 6–8 weeks old were induced to superovulate with an intraperitoneal injection of 7.5 IU equine chorionic gonadotropin (eCG, Folligon, Intervet, The Netherlands), followed 48 h later, by 5 IU of human chorionic gonadotropin (hCG). Immediately after hCG administration, females were paired with adult intact mice of the same genetic background in a individual cage overnight. All animal experiments were approved by our Institutional Review Board according to the European and Spanish legislation.
One-cell embryos were collected from the ampulla of females which were euthanized by cervical dislocation, 20 h post-hCG administration. Cumulus cells were removed by exposure to hyaluronidase as described  and zygotes were washed in M2 medium and cultured in Potassium (K+) Simplex Optimized Medium (KSOM) microdrops  covered with mineral oil at 37°C and 5% CO2 until the morula stage. Morulae embryos developed in vivo were collected 66–78 h post-hCG administration by flushing both the oviduct and the upper part of the uterine horn with M2 medium.
Three temperatures (4°C, 15°C and 37°C) and two media (KSOM and KSOM-H) were chosen. Additionally, two storage lengths (24 and 48 hours) were studied in order to assess the effect of storage on in vitro development. Buffered media (KSOM-H) for embryo storage was prepared by adding HEPES (20 mM) to KSOM, maintaining bicarbonate at the original concentration of 25 mM. KSOM composition is shown at 23. Both media were prepared every 2 weeks and filtered through 0.22 μm pore and stored at 4°C until use.
In experiment 3 (in vitro study), thirteen B6CBAF1 females and six B6CBAF1 males were used: 1-cell embryos collected on day 0.5 were placed in culture for 48–50 hours until reaching the morula stage. As described for the two previous experiments, groups of 20–30 embryos were placed in cryotubes filled with pre-equilibrated (24 h before) KSOM (5 ml) or KSOM-H and randomly assigned to one of the three storage conditions selected: 4°C, 15°C or 37°C. 24 hours later, embryos were recovered from each tube and placed in culture. For this experiment, a group of non-stored embryos obtained in the same way was set in culture as a control group for development.
pH determination of holding media under the different storage conditions tested
In order to evaluate the pH fluctuations that might occur in equilibrated and buffered media used for embryo storage, pH measurements were performed on samples in triplicate, maintained under experimental storage conditions at 0, 90, 180 and 300 minutes, 24 h, 48 h and 72 h. for each treatment. Mean values were used as pH measurement at each time point.
In vitroevaluation of viability of stored embryos
In vitro viability was evaluated in three different groups: in vivo collected morulae stored for 24 hours (experiment 1) or 48 hours (experiment 2) and morulae obtained from 1-cell embryos after in vitro culture and then stored for 24 hours (experiment 3). To perform these experiments, seventy-five B6CBAF1 females were used as embryo donors, seventy-three CD-1 females as surrogate mothers; ten B6CBAF1 males and ten CD1 males were used to establish matings. In all cases, embryos were randomly distributed into the six combinations of media and temperature described, plus into a control group of cultured embryos without previous storage. Two parameters were determined: percentage of development to blastocyst, and total cell count. To determine the in vitro viability based on development to blastocyst, embryos were recovered after storage from cryotubes and placed in pre-equilibrated KSOM microdrops. After incubation at 37°C and 5% CO2 for 24–36 h, the number of expanded blastocysts was recorded.
The total cell number of blastocyst which had developed under the different storage conditions and in control groups was determined by nuclei staining. Embryos were placed in M2 medium drops supplemented with Hoechst 33342 (1 μg/ml) and allowed to stain during 30 minutes at 37°C. Blastocysts were placed on a glass slide, sealed with a cover slide and observed under a Nikon Optiphot-2 fluorescence microscope with an appropriate filter set (Blue: 450-490 nm excitation filter, 515 nm barrier filter).
In vivoevaluation of viability of stored embryos
Embryos stored under the different conditions were, immediately after the storage period, surgically transferred to surrogate mothers in order to establish their in vivo viability. 15 to 20 embryos recovered from the different treatments were transferred into the left oviduct of 0.5 pseudopregnant CD-1 females (Harlan), plugged by vasectomised males. At day 14 of gestation, recipients were euthanized and uterine horns were isolated to record the number of living fetuses, resorptions and the total number of implantations.
Total cell counts of blastocysts developed after morulae ( in vivo developed) storage during 24 hours
Number of blastocysts analysed
Total cell (N° of cells)
59.26 ± 2.86 a
40.77 ± 1.96 b
48.94 ± 2.67 b
40.24 ± 2.05 b
44.58 ± 2.12 b
46.10 ± 3.11 b
50.46 ± 3.93 b
Our aim was to assess viability of embryos stored in different conditions that could be easily used for transport without requirement of specific containers or specialized recovery techniques. Temperatures were selected to include those whereat embryos could be in a latent stage (4°C) , or at a physiological developmental temperature (37°C) [13, 14]. An additional temperature was selected whereat metabolism could be retarded and easily maintained without complex equipments (15°). These temperatures were combined with 2 different media: KSOMeq and KSOM-H. Three different studies were designed to achieve this goal. In the first study (Figure 1) we assessed in vitro viability after storage in three experiments: In experiment 1, freshly collected morulae were stored in different conditions for 24 hours. In experiment 2, the same storage conditions were tested for 48 hours, and in experiment 3, embryos were collected at the 1-cell stage, cultured in vitro until the morula stage and then submitted to the same storage conditions for 24 hours as in experiment 1. In vitro viability was determined based on percentage of development in culture and assessing total cell number.
In the second study, in vivo viability of stored embryos was evaluated based on the number of live implantations and resorptions after being transferred into foster mothers. In vivo assessment of viability was circumscribed to embryos stored for 24 h; either freshly collected morulae or morulae obtained after in vitro culture of embryos collected at the 1-cell stage, since in those groups, conditions were represented that, in vitro, were either detrimental or did not affect embryo development.
The third study explored the effect of storage conditions on expression of some genes closely related to early embryo development. To determine these levels of gene expression, we chose embryos obtained in vivo stored for 48 h under the different conditions studied. We selected this time condition in order to examine gene expression after storage in those combinations of temperature and media in which a relevant effect was assessed. Correlation between gene expression and physiological aspects (embryo development in culture) allows for defining new molecular aspects of embryo physiology.
Statistical analyses were performed using SigmaStat version 3.1.1 software (Jandel Scientific, San Rafael, CA). Data is given as the mean ± S.E.M. Comparison of the differences between means for each treatment were done using ANOVA followed by Student-Newman-Kleus post hoc test.
In vitrodevelopment after storage for 24 or 48 hours
Total cell number
Correlation between the cell number in the blastocyst, and implantation ability was reported several years ago . However, this parameter correlates poorly with morphology, a visual character widely used as a non-invasive marker for embryo quality. For this reason, morphological criterion should be reinforced by other parameters . In addition to morphology, we analyzed the total cell number of morulae developed in vivo subjected to storage during 24 hours. As a result of this study, we observed a significant cell number decrease in all treatments in comparison with the control group (Table 1), but no differences were found when we compared this parameter within the different experimental conditions tested.
Total cell count of blastocysts developed after morulae ( in vivo developed) storage during 48 hours
Number of blastocysts analysed
Total cell (N° of cells)
69.43 ± 3.19 a
64.67 ± 1.97 a
70.05 ± 2.80 a
41.14 ± 2.66 b
43.30 ± 2.12 b
52.97 ± 1.39 c
38.15 ± 1.47 b
Total cell counts of blastocysts developed after morulae ( in vitro developed) storage during 24 hours
Number of blastocysts analysed
Total cell (N° of cells)
54.73 ± 1.74a
40.00 ± 1.98b
44.88 ± 2.38 b
43.63 ± 1.85 b
32.61 ± 3.08 c
52.39 ± 2.97a
32.83 ± 2.96 c
In vitro developed morulae showed a significant decrease in cell number in all treatments, except in KSOM at 37°C, with the highest decrease found in KSOM-H at 15°C and at 37°C. This fact indicates that media alkalinization proved to be less harmful than HEPES presence, since we found a significant difference in this temperature (52.39 vs 32.83 (KSOM at 37°C vs KSOM-H at 37°C)). This effect was not present in those embryos collected as morulae and subjected to the same storage conditions as those described in experiment 1 (Table 3). This suggests that the coincidence of HEPES presence was necessary with other detrimental conditions in order to produce a measurable negative effect.
In vivoviability of stored embryos: analysis of implantations and fetuses
Number of fetuses, resoprtions and total number of implantations after embryo transfer of morulae ( in vivo developed) stored during 24 hours
Number of experiments
Total implantations (%)
41 (58.48 ± 7.76) a
11 (15.90 ± 4.98)nd
52 (74.37 ± 6.83)nd
38 (49.59 ± 9.57) a
16 (21.15 ± 8.23)
54 (70.75 ± 13.63)
30 (42.92 ± 5.55) a
15 (32.34 ± 7.41)
45 (75.26 ± 6.18)
33 (41.05 ± 4.02) a
31 (38.70 ± 4.63)
64 (79.75 ± 5.31)
47 (66.29 ± 2.79) a
14 (20.19 ± 3.57)
61 (86.47 ± 4.72)
41 (52.95 ± 9.97) a
18 (22.16 ± 6.41)
59 (75.11 ± 10.62)
44 (56.21 ± 6.28) a
19 (24.01 ± 10.22)
63 (80.23 ± 5.81)
Number of fetuses, resoprtions and total number of implantations after embryo transfer of morulae ( in vitro developed) stored during 24 hours
Number of experiments
Total implantations (%)
24 (30.6 ± 2.7)a
19 (23.6 ± 4.09)a
43 (57.5 ± 5.74)n.d.
31 (32.8 ± 6.38)a
25 (29.8 ± 8.96)ab
56 (62.59 ± 5.78)
21 (29.5 ± 6.38)a
26 (34.5 ± 5.27)ab
47 (64.07 ± 6.90)
11 (13.6 ± 3.99)ab
35 (40.4 ± 6.80)ab
46 (53.96 ± 8.62)
4 (4.67 ± 2.34)b
47 (63.9 ± 13.4)b
51 (69.94 ± 12.89)
12 (16.4 ± 5.60)ab
40 (45.2 ± 6.35)ab
52 (60.49 ± 9.15)
17 (17.0 ± 3.76)ab
36 (37.8 ± 8.40)ab
53 (54.81 ± 9.48)
Transcription analysis by real time PCR
Details of primers used for RT-PCR
MGI official name
Primers sequence (5′– 3′)
Gene bank accesion n°
BCL2-associated X protein
Murine endogenous retovirus-L
Telomeric repeat binding factor 1
Gap junction membrane channel protein alpha 1
POU domain, class 5, transcription factor 1
Retrotransposon expression, frequently impaired in stressful embryo situations, was analysed measuring mRNA levels of Erv4 and Iap genes. Erv4 was significantly upregulated in KSOMeq and KSOM-H at 15°C, Furthermore, Iap showed a marked increase in mRNA levels in embryos kept in KSOM-H at 5°C when compared to controls. These changes in retrotransposon transcription indicate some alterations in the epigenetic control of their expression. While no effect was detected in the expression of reverse transcriptase for telomerase (Tert), Gaj1 (Connexin 43) showed a decrease in levels of transcripts at 15° and 37°, both in KSOMeq and in KSOM-H. This situation could have detrimental effects on the implantation of stored embryos, advising against the use of these temperature conditions for embryo transport (Figure 6D).
Nanog transcript levels decreased in all treatments compared with the control group, but the effect was especially remarkable in KSOM and KSOM-H at 15° and 37°C. A similar pattern had Pou5F1 (Oct 3/4), decreasing transcripts in KSOM at 15°C and both KSOM as KSOM-H at 37°C (Figure 6C).
Gapdh is a gene involved in metabolic functions, such as in transcription and programmed cell death. In this case, it had a slightly significative tendency to be downregulated at higher temperatures. KSOM at 15C and KSOM-H at 37°C were the groups in which downregulation was more evident (Figure 6B).
Holding medium pH fluctuation during storage
Since our intention was to test standard recipients within the reach of any laboratory, we performed the study in cryotubes. These recipients close tightly and provide a theoretical way to isolate the growth media in which embryos could be stored. Analysis of pH revealed a significant difference in pH values of KSOMeq at 37°C in comparison with the the rest of treatments. These findings demonstrated that cryotubes allow CO2 difussion into the air and a concomitant basification that was not buffered by the presence of HEPES as in KSOM-H, and that this diffusion was potentiated by temperature . More efficient ways to preserve storage environment have been reported , that should be considered as an alternative if transport has to take place under such conditions.
Shipment of embryos is a good alternative to the exchange of live animals for several reasons. The intrinsic sterility of embryos surrounded by an intact zona pellucida obviates differences in the microbiologic levels of donor and recipient laboratories , . Transport of embryos also has the advantage of avoiding stressful conditions that may affect animals for several days. In addition, use of embryos when dealing with genetically modified animals avoids any possibility of accidental escape, an important issue in risk assessments. Embryos of different mammalian species have been successfully transported in frozen stage [21–23]. Cryopreserved embryos are a reliable method and they can reach any remote location in the world; conversely, fresh embryos remain viable for a limited amount of time and they require careful coordination between sending and receiving facilities. However, they allow the use of simple containers instead of dry shippers and, what is more important: they do not require cryopreservation skills to correctly freeze and thaw embryos.
These advantages have motivated several studies to set up non-frozen transport conditions [24–26]. In most cases, they were circumscribed to the 2-cell stage. The reason to choose this specific developmental stage may rely on providing the more extended length of in vitro culture for positively fertilized embryos. However, this stage poses a risk in certain strains, since 2-cell block mostly appears linked to suboptimal culture conditions [7, 27–29].
We wanted to define more universal conditions by exploring different temperatures and the role of pH, comparing an equilibrated versus a HEPES buffered medium. We also chose morula stage, a less restrictive stage since the 2-cell block has already been overcome and there are still 24–48 hours of autonomy. In addition, we explored the feasibility of collecting embryos at 1-cell stage and culturing them to morulae before storage, since collecting embryos from the ampulla is consistently easier than flushing them from the oviduct, and our aim was to define the most accessible way to ship embryos under non-frozen conditions.
Our results in the first experiment demonstrate that in vitro development after 24 hours of storage under the conditions described is not compromised. Our findings showed that, even though there was a significant decrease of cell counts in all treatments with respect to the control group, this did not compromise in vivo viability after transfer. Somehow, the certain delay in development due to storage, which motivates the decrease in total cell numbers in stored embryos, was easily overcome when they were placed in a permissive maternal environment, such as a 0.5 recipient. This demonstrates that successful transfer of morulae after 24 h of storage can be achieved in any of the media or temperature assayed without compromising in vivo viability. Previous studies have focused on the transport of 2-cell embryos, either thawed after cryopreservation,  or obtained from in vitro fertilization , achieving 44% in vivo development for embryos stored at 4°C for 48 hours. We obtained higher developmental levels under all the conditions tested, but in our study, embryo transfer was performed after 24 hours of storage. Given the significant decrease of in vitro viability observed when comparing 24 to 48 hours of storage, and the detrimental effect of in vitro embryo culture previous to being subjected to storage conditions, we can assume that for longer storage periods, 4°C is more advisable. At 15°C or 37°C, the induced gene deregulation and the decrease in cell count may not be compensated by the maternal environment after transfer, as occurred with 24 h stored embryos.
The in vivo and in vitro alterations observed after the storage 48 h may be a consequence of the misregulation of genes with an important role in embryo development. For example, the low percentage of blastocyst development after storage at 15°C can be related to the increase in deregulation in Bax/Bcl-2 ratio and the increase in the retrotrasposon expression observed in this group, since Bcl2 Bax ratio has been considered a marker of viability or apoptosis in bovine embryos .
The cell number decrease observed both in the 15°C and 37°C groups could be related to the decrease in Nanog and Oct3/4 expression. The expression patterns we found might also suggest a decrease in the pluripotency ability of ICM cells and, to some extent, may be responsible for the failure in postimplantation development found after transfer of stored embryos at 15°C [32, 33]. Expression patterns in the group of embryos stored at 4°C, were more similar to the patterns observed in the control group, which could also explain the higher percentage of living fetuses obtained after transfer of embryos collected at 1-cell and in vitro cultured prior to storage.
Although several studies have demonstrated embryo ability to develop after long periods under non-physiological conditions [6, 34] , we did not intend to analyze culture conditions. Differences between those analyses and the findings presented here are relevant. We focused on defining a way to store embryos from a practical point of view; for this reason, we compared several temperatures instead of focusing only on one. On the other hand, since long- term transport is successfully achieved with frozen embryos, we focused on short-term conditions. Nowadays, most courier transports deliver in 24–48 hours. Therefore the aim of our study was not to examine in depth what the limit for survival of an embryo is under different conditions, but rather if any of the conditions analysed could be addressed as the optimal one for short-term shipment.
Standard embryo holding media, such as M-2 substitute bicarbonate with HEPES, are useful in order to stabilize pH when embryos are handled; however, bicarbonate is necessary for the optimal development of embryos  while being kept at 4°C. Buffering is not the only role of NaHCO3, since CO2 is required for in vitro embryo development. Carbon from external CO2 is fixed by the embryo and used for various metabolic processes [35, 36]. Some authors consider that buffer mixtures as HEPES-MOPS together with 25 mM NaHCO3 are enough to maintain pH .
With this in mind, in our experimental design, bicarbonate was not removed from the medium. KSOM-H consisted of the same standard KSOM medium with 25 mM sodium bicarbonate, supplemented with HEPES, in order to provide pH buffering ability without reducing the amount of available bicarbonate.
The volume in which embryos were contained is an important parameter. Mouse embryos are sensitive to osmolarity increase ; due to this circumstance, and so as to avoid evaporation during storage time, a large volume for the medium was chosen. In addition, pH buffering is more accurate in large volumes, especially in KSOMeq. For this reason, storage conditions should be considered as a whole, since volume may influence gas interchange and pH fluctuation.
In this study, most treatments implied a significant reduction in cell numbers in comparison with the control, but these differences did not affect implantation ability . Reduced cell numbers per embryo do not always indicate a decline in the subsequent in vitro or in vivo developmental capacity of embryos, as some authors have reported in other processes, such as cryopreservation . Some theories have been proposed, but essentially, embryos with reduced numbers of cells are still able to give rise to fetuses [40–42]. Nowadays, is not fully understood what the limit is to this situation. It is proposed that ICM cells have an important role in the three germ layer formation of the embryo. If too small, ICM is present, so embryonic endoderm cannot be formed. Although only the embryonic ectoderm gives rise to the embryo proper, the interactions between the three germ layers are necessary for full embryonic and fetal development.
Our aim was to define the easiest way possible to obtain and store embryos for a short time, eliminating the need for cryopreservation. For this reason, in our study, we also tested the feasibility of using morulae obtained after in vitro culture of embryos collected at the 1-cell stage. One cell embryo collection is technically easy, and in vitro culture avoids the necessity of correct identification of fertilized embryos, which requires a certain skill. However, this approach proved to be less efficient. Even though live fetuses were obtained from all storage conditions, there was a sensitive increase in the number of resorptions. This demonstrated that embryos had a compromised viability; they could be implanted, but were incapable of further development. Our results regarding cell counts somehow illustrate that situation: morulae obtained in vitro had a lower total cell number in comparison with the numbers observed on in vivo collected morulae. Even though embryos with reduced cell numbers are able to implant and develop, a low cell count has been associated with lower developmental ability .
Our results show that morula is a very permissive developmental stage for 24 h embryo storage, allowing different temperatures and media conditions without detrimental effects on in vivo viability after transfer. Even though previous in vitro culture seriously compromises embryo viability, this effect is more dramatic when storing under suboptimal conditions, such as 15°C where deregulation of important genes became highly significant. But even under such conditions, it was possible to obtain live fetuses after transfer. Since none of the 24 h storage conditions tested showed a significant improvement or a deleterious effect, there is no scientific basis to choose one or other. Practical preferences are a valid criterium to decide on a given temperature. However, if it is suspected a certain delay in the process or previous culture of embryos is necessary, our results based on the higher implantation rates found, suggest that 4°C provides a safer margin.
In conclusion, both KSOMeq and KSOM-H may be equally used for embryo storage, and several temperature conditions allow for good in vitro and in vivo survival. Some of these storage conditions can substitute freezing in order to maintain embryo viability for 24–48 hours, offering a good and technically less demanding alternative for embryo exchanges.
This work was performed as part of, and financed by, Project AGL2004-00332. JDH received a Ph D. grant from the INIA (Ministry of Science and Innovation). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its unit of Information Resources for Research (URICI). Language correction has been performed by the Oficina de Asesoría Lingüística of the Autónoma University Foundation.
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