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BOR - Papers in Press, published online ahead of print August 25, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025916
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BIOLOGY OF REPRODUCTION 71, 2048–2055 (2004)
DOI: 10.1095/biolreprod.103.025916
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Post Hatching Development: a Novel System for Extended in Vitro Culture of Bovine Embryos

Daniela O. Brandão1,2,3,5, Poul Maddox-Hyttel4, Peter Løvendahl3, Rodolfo Rumpf3,5, David Stringfellow6, and Henrik Callesen3

Laboratório de Reprodução Animal I,2 Embrapa Recursos Genéticos e Biotecnologia, C.P. 02372 Brasília, DF, Brazil Section of Reproductive Biology,3 Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark Department of Anatomy and Physiology,4 Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark Laboratório de Biologia Molecular,5 Departamento de Biologia Celular, Universidade de Brasília, CEP 70910-900 Brasília, DF, Brazil Department of Pathobiology,6 College of Veterinary Medicine, Auburn University, Alabama 36849-5519


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although acceptable rates of blastocyst formation are achieved with in vitro production of bovine embryos, several problems still compromise the subsequent development of the fetus and newborn, especially in embryos originating from somatic cell nuclear transfer. Routinely, the potential development of a bovine conceptus is predicted either on blastocyst quality or on various parameters related to the embryonic-fetal development in a foster mother. These methods are either imprecise or costly, highlighting the need for more reliable and practical methods to evaluate early embryonic development and differentiation. Thus, our aim was to improve the in vitro culture of embryos post hatching and to define a stable and repeatable system to monitor the development of bovine embryos. For that, in vitro–derived embryos were cultured in agarose gel tunnels in a modified culture medium (SOFaaci within 10% fetal bovine serum and 27.7 mM glucose). Daily monitoring of embryo length revealed that 56%–67% of the embryos in culture showed rapid growth and elongated until Day 13. Electron microscopy of elongated embryos at Day 14 confirmed successful localization of differentiated cells forming the trophoblast and hypoblast, with the definition of the Rauber layer. In conclusion, a stable culture system of post hatching embryos was first defined and can be used as a model for rapid growth, elongation, and initial differentiation of bovine post hatching embryos produced entirely in vitro.

bovine, conceptus, developmental biology, early development, embryo, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The high number of abnormalities and pregnancy losses found in fetuses and offspring produced by somatic cell nuclear transfer [1–5] and also found in lower frequency with in vitro–produced embryos [6–9] suggests that blastocyst evaluation per se is not an ideal parameter for predicting subsequent developmental competence [10]. Even when new cloning methods achieve high blastocysts rates in the bovine, normal gestation and birth rates are still low, ranging from 2% to 5% [2, 11, 12]. Several developmental problems such as early embryonic or fetal mortality [3, 13], fetal anomalies, high birth weight [14], and placental alterations [2–4] are frequently reported. These findings highlight the need for new methods to monitor and investigate embryo progress, rather than the conventional limits of embryo morphology on Days 7 or 8, especially for cloned embryos. For that, prolonged culture in vitro would be a more practical and useful approach than in vivo development following embryo transfer.

In vitro cultures of post hatching embryos have been described in primate [15, 16]; rat [17, 18]; rabbit [19]; marsupial [20]; and hamster [21]. Such an approach is often used as a monitoring parameter in studies on the effects of toxic components or genetic influence on early embryo development. However, only in vivo–developed and flushed embryos are used and usually for a restricted short term in vitro culture.

The possibility of the in vitro culture of bovine embryos post hatching was first observed by Stringfellow and Thompson [22] who achieved embryo elongation with in vivo–produced and flushed bovine embryos that were placed inside agarose gel tunnels and cultured in vitro. Further progress was reported by Vajta et al. [23] when in vitro–produced embryos cultured inside agar gel tunnels achieved not only elongation, but also a high survival rate in long-term culture until Day 26 with initial signs of differentiation. To improve the developmental capacity, in vitro–produced embryos between Days 11 and 15 were cultured in vitro in wells under different substrates and atmospheric conditions [24–26]. However, most of the basic experiments testing culture conditions and composition were performed with the culture of embryos growing freely into traditional culture dishes. In these conditions, the embryos either attach to the bottom or grow in rounded form, and the elongation was never spontaneously found. In spite of the occurrence of an incomplete hypoblast layer, extensive degeneration has occurred and the complete embryonic disc was not identified. All of those previous works are the initial investigative steps and the observation of the phenomena of elongation in vitro. However, there was not a culture system established or defined patterns for post hatching development of bovine embryos in vitro.

Based on these first reports, our goal was to improve the technical difficulties in establishing an in vitro system allowing post hatching development of bovine embryos in vitro with high and stable rates. We would furthermore do this using a large material in which some of the factors of importance for the function of the system could be investigated, and also to document the normal signs of early development of the embryos using ultrastructural investigation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.

In Vitro Embryo Production

The method used for in vitro embryo production was previously described elsewhere [27]. Briefly, oocytes were aspirated from slaughterhouse-derived ovaries and selected and matured for 24 h in four-well dishes (Nunc, Roskilde, Denmark). Each well contained 400 µl of bicarbonate buffered TCM-199 medium (Gibco BRL, Paisley, UK) supplemented with 15% cattle serum (CS; Danish Veterinary Institute, Copenhagen, Denmark) and 10 IU/ml eCG and 5 IU/ml hCG (Suigonan Vet; Intervet, Skovlunde, Denmark) under mineral oil at 38.5°C in 5% CO2 in humidified air. Fertilization was performed in modified Tyrode medium [28]. After 22 h, cumulus cells were removed by vortexing, and presumptive zygotes were transferred to culture in 400 µl of synthetic oviduct fluid medium with amino acids, citrate, and inositol (SOFaaci) [27] supplemented with antibiotics (gentamicin sulfate, 10 mg/ml) and 5% CS and incubated at 38.5°C in 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity. On Day 7, rates of blastocysts (i.e., early, full, expanded, hatching, or hatched blastocysts) were recorded. On Day 8 all degenerated structures or nonhatched blastocysts were removed from the culture dish. Embryos were kept in the same well from Day 0 (day of fertilization) until Day 11.

Post Hatching Development (PHD) System

The PHD system consisted of tunnels produced in agar gel and covered with culture medium. Glass capillaries of 120 mm length x 1.2 mm width (World Precision Instruments Inc., Sarasota, FL) were shortened to 65 mm pieces, and one of the open ends was then closed by melting the glass over a gas flame. Eight capillaries oriented in parallel spaced 2 mm apart from each other were fixed at the open end with an autoclave tape (Selefa Trade, 131250-13, VWR International, Albertslund, Denmark), and the resulting "comb" was immersed into 70% ethanol until used.

Agarose gels were produced by dissolving 2.4% of low–melting-point agarose (15517-022, Gibco BRL, Gaithersburg, MD) in PBS. The solution was autoclaved and cooled to 40°C. All subsequent steps were performed under sterile conditions. The closed ends of two glass combs were briefly sterilized with a flame before they were placed in opposite directions in a Petri dish (60 x 15 mm; Nunc, Roskilde, Denmark), with the closed ends placed in the bottom and the tape side placed on the border of the Petri dish. Fetal bovine serum (FBS; Biochrome AG, Berlin, Germany) was added to the agar solutions in a concentration of 4.5% or 9% (Agar4.5 and Agar9, respectively). Immediately after serum supplementation, 10 ml of the agarose gel solution was poured over the combs in each Petri dish. The dish was placed on ice bags for 10 min for rapid solidification of the gel without excessive evaporation. Subsequently, 2 ml of PHD medium (SOFaaci supplemented with 0.5% glucose and 10% FBS) was poured on the gel surface and the combs were slowly removed, forming diagonal tunnels of 20 mm length x 1.2 mm width filled with medium and free of air bubbles. A sterile blade was then used to cut the tunnels to 15 mm length and remove excess gel before only one group of eight tunnels was placed in each Petri dish. Using a narrowed and curved Pasteur pipette, the lumen of the tunnels were flushed with the medium once a day and during two subsequent days, and the dish was then kept in 10 ml of PHD medium in the incubator at 38.5°C in 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity until utilization. The final dish, containing the gel and the PHD medium ready to use, was designated as the PHD dish.

Embryo Culture in the PHD System

On Day 9, some hatched blastocysts were removed for transmission electron microscopy analysis and the remaining blastocysts were kept in culture. In sequence, 400 µl of PHD medium was slowly added and mixed in each well, increasing the final volume to 800 µl. On Day 11, the embryos were evaluated under stereomicroscope considering three parameters: diameter, trophoblast layer, and inner cell mass. The embryos were scored as Quality I, ≥1.0 mm diameter, clear trophoblast, compact inner cell mass; Quality II, ≥0.5 mm diameter, few dark spots in the trophoblast, slightly losing cells in the inner cell mass; or Quality III, <0.5 mm diameter, numerous dark spots in the trophoblast, spread inner cell mass. Embryos showing visible degeneration were discarded. Using a 100 µl pipette tip with the end cut off with a sterile blade, Day 11 embryos were removed from the culture dishes in groups of eight and placed in the prepared PHD dish. With a closed and bowled tip of a Pasteur pipette, each embryo was gently pushed approximately 0.5 mm inside the lumen of one gel tunnel. The loaded embryos remained in the PHD culture until Day 14 or 15, depending on the treatment. The culture was performed at 38.5°C in 5% CO2, 5% O2, and 90% N2 atmosphere.

Evaluation of Embryos

To evaluate the impact of short-term changing temperature and pH on the subsequent development of the embryos growing in the PHD system, half of the PHD dishes were left undisturbed in the incubator by Day 12. The other half of the PHD dishes in culture were randomly removed from the incubator to measure embryo length inside their tunnels with an ocular micrometer eyepiece. On Days 13 and 14, all PHD dishes were removed from the incubator and the length of all embryos were measured. Embryos were classified as "elongating" when they reached ≥1.3 mm length. During the culture period, three patterns of growth were observed and defined: 1) degenerated, embryos that degenerated or reduced in size; 2) discontinuous, embryos presenting days with elongation combined with days without any elongation; and 3) continuous, embryos presenting progressive elongation daily. On Day 14, one third of the PHD dishes randomly selected were removed from culture and the gels were placed into another Petri dish with PBS at 38°C. Then, each embryo was aspirated from its gel tunnel with a 3-ml plastic Pasteur pipette (Elkay Eireann, Galway, Ireland). From one end of each elongated embryo, a biopsy of the trophoblast layer (1 mm in length) was taken with a sterile blade and placed in an Eppendorf tube containing 20 µl PCR buffer and frozen at –20°C for gender diagnosis. Subsequently, the same Day 14 embryos were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h at 4°C for transmission electron microscopy [10]. The Day 14 embryos were picked for morphological evaluation to compare with the in vivo parameters found in the literature [10, 29–31]. The embryos in the remaining PHD dishes were length-measured on Day 15 again. For evaluating embryo elongation in the PHD culture beyond Day 15, a few embryos were kept in culture for another 3–5 days.

Processing for Transmission Electron Microscopy

Embryos (Day 9, n = 3; Day 14, n = 4) were fixed in 3% glutaraldehyde in 0.1 M Na-phosphate buffer (pH 7.2) and kept at 4°C. Immediately before further processing, the Day 14 embryos were placed in a Petri dish containing 0.1 M PBS, and the region around the potentially developing epiblasts was isolated under the stereomicroscope. Subsequently, the Day 9 embryos and the isolated epiblasts of Day 14 embryos were washed in buffer, postfixed in 1% OsO4 in 0.1 M Na-phosphate buffer, embedded in Epon, and serially sectioned into semithin sections (2 µm). The sections were then stained with basic toluidine blue and evaluated by bright field light microscopy. Selected semithin sections presenting features of the development of the epiblasts were re-embedded [32] and processed for ultrathin sectioning (70 nm). The ultrathin sections were contrasted with uranyl acetate and lead citrate and examined on a Philips CM100 transmission electron microscope.

Gender Diagnosis

The gender diagnosis was performed as previously described by Roschlau et al. [33] for Day 7 embryos and adapted for Day 14 embryos in the present experiment. For DNA release, Proteinase-K (Merck 1 mg/ ml) was added to the PCR buffer containing the biopsies and incubated at 60°C for 1 h. An aliquot of 1 µl lysate was used for PCR with bovine Y-chromosome–specific primers derived from the DNA sequence bov 97 [34]. Amplification was performed in a total volume of 20 µl. The reaction mixture contained Stoffel Buffer, 200 µM each dNTP, 10 pmol of each sexing primer, 4 mM MgCl2, and 1.5 U of DNA polymerase Stoffel fragment (Perkin Elmer Inc., Boston, MA).

After 30 cycles the male-specific fragment of 157 bp was visualized in 2% agarose. Samples without a male sequence were retested in a multiplex PCR with additional 1 pmol of control primers derived from a bovine highly repetitive sequence [35]. Samples carrying only the 220 bp bovine fragment were considered as female, and those with both fragments as males.

Statistical Analyses

For the whole experiment, a stepwise analysis was conducted, considering that embryos were leaving the system at different stages. The analysis of the further steps were conditional to survival in all of the prior steps. Treatment or grouping effects on rates of survival to any next step were tested by {chi}2. Quantitative measures (length of embryo on Days 13, 14, and 15 and elongation rates) were analyzed by a mixed model using SAS software (Mixed Procedure; SAS Institute Inc., Cary, NC). The model contained fixed factors of experimental treatments: gel source (Agar4.5/ Agar9), embryo quality group (1/2/3), measurement on Day 12 (yes/no), and interactions between those. Variances from effect of PHD dishes and gel differences between replicates were included as random terms together with the residual variance. The pattern of the embryo's growth in the system was assessed quantitatively by attributing points to growth patterns for each of Days 13, 14, and 15. A score of 3 points was given to the category including embryos consistently elongating over the 3 days; 2 points for embryos with irregular development or getting stuck; and 1 point to embryos not developing at all or degenerating until Day 15. The survival score was analyzed using the model for quantitative traits. The embryos and gels that were submitted to histology on Day 14 had no measurements on Day 15 and were omitted from further analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
On Day 7, 1.116 embryos (43% ± 6% of the total number of oocytes subjected to maturation, n = 12 replicates) were classified as early to hatched blastocysts, whereas on Day 8 this number had increased to 1.234 (48% of total oocytes). From these, 357 embryos (32% ± 5% of the Day 8 embryos, n = 12) were considered to be viable on Day 11, and 304 of them were successfully loaded to the PHD culture. The main reason of embryo loss during loading procedure was the collapse of embryos due to either large size (>1.2 mm) or their low quality. Among the 304 embryos included in the PHD culture, 22% were classified as Quality 1, 59% as Quality 2, and 19% as Quality 3.

Embryos Initiating Elongation

In total, 170 embryos initiated elongation (56% of 304 embryos loaded). The percentage of embryos initiating elongation was higher in the Agar9 than in Agar4.5 gels when seen across quality embryos (67% versus 48%; P < 0.05), and within quality groups (Table 1). In both types of gels the percentage of embryos initiating elongation decreased with lower quality score (Table 1).


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  		TABLE 1. Bovine embryos with different qualities (Day 11) initiating elongation in the PHD system, using agarose gel supplemented with 4.5% or 9% fetal bovine serum (FBS) and cultured in vitro until Day 15 of development

Embryo Length

Only embryos that initiated elongation (n = 170) were evaluated for embryo length. Average length of the embryos on different days of PHD culture is presented in Table 2, independent of the elongation pattern. The length of the embryos increased from Days 12 to 15, but more so in the best quality embryos than in those of lesser grades (Table 2).


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  		TABLE 2. Average length (mm) of elongating bovine embryos with different qualities on Day 12 (n = 65), Day 13 (n = 170), Day 14 (n = 170), and Day 15 (n = 105 after removing 65 embryos on Day 14) of development when cultured in the PHD system

Although not subjected to statistical analysis, some embryos were kept in the PHD system beyond Day 15 for evaluating potential embryo growth. The longest embryos reached 10.1 mm on Day 17 (Quality I embryo) and 12.5 mm on Day 19 (Quality II embryo).

Elongation Pattern

Apart from the embryos that were removed from the PHD culture on Day 14 for gender diagnosis and fixation, a total of 105 embryos reached Day 15 and were used to illustrate the elongation pattern through the culture period (Day 12 to Day 15). From these, 34% (n = 36) degenerated within the culture period, 47% (n = 45) showed discontinuous elongation, and 23% (n = 24) showed continuous elongation until Day 15. However, the elongation pattern differed depending on the embryo quality group (Fig. 1).



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  		FIG. 1. Elongation patterns of bovine embryos of different qualities cultured in the PHD system between Days 12 and 15 of development. Quality I (white bars), >1.0 mm diameter, clear trophoblast, compact inner cell mass; Quality II (gray bars), >0.5 mm diameter, few dark spots in the trophoblast, slightly losing cells in the inner cell mass; or Quality III (black bars), <0.5 mm diameter, numerous dark spots in the trophoblast, spread inner cell mass. Values are least square means. *Denote significant difference ({chi}2 test; P < 0.05)

The effect of measurement on Day 12 had a significant effect in some embryo quality groups. When the embryos were measured, the frequency of embryos presenting continuous elongation was lower compared with the ones that degenerated or that presented discontinuous elongation (12.3% [n = 8], 47.7% [n = 31], and 40.0% [n = 26], respectively; P < 0.05). However, when embryos were not measured, the percentage of embryos that degenerated was lower than the embryos that showed discontinuous or continuous elongation (12.5% [n = 5], 47.5% [n = 19], and 40.0% [n = 16], respectively; P < 0.05). Results are shown in Table 3 and Figure 2.


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  		TABLE 3. Effect of measurement on Day 12 on subsequent elongation pattern until Day 15 (degenerate, discontinuous, or continuous) of bovine embryos cultured in the PHD system



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  		FIG. 2. Elongation patterns of bovine embryos of different qualities cultured in the PHD system between Days 12 and 15 of development when not measured (dotted) or measured on Day 12 (lines). A) Elongation pattern for Quality I embryos. B) Elongation pattern for Quality II embryos. C) Elongation pattern for Quality III embryos. Quality I: ≥1.0 mm diameter, clear trophoblast, compact inner cell mass. Quality II: ≥0.5 mm diameter, few dark spots in the trophoblast, slightly losing cells in the inner cell mass. Quality III: <0.5 mm diameter, numerous dark spots in the trophoblast, spread inner cell mass. *Denote significant differences within elongation pattern group ({chi}2 test; P < 0.05)

It was also observed that the trophoblast established an intimate contact with the surrounding tunnel, extending along any failure or crack present in the agar gel.

Transmission Electron Microscopy Analysis

All Day 9 embryos were spherical in shape and displayed a well-defined inner cell mass. The innermost cell layer of the inner cell mass had in all cases delaminated into a flat layer of hypoblast cells. However, the hypoblast layer was only seen extending in relation to the inner cell mass, but not on the inside of the trophoblast layer. The remaining cells of the inner cell mass are at this point referred to as epiblasts. The epiblasts displayed abundant signs of degeneration in the form of apoptosis (nuclear compaction and formation of apoptotic bodies) and necrosis (swelling and disintegration). In two out of three embryos, the trophoblast cells, clearly recognizable on their abundant apical microvilli covering, formed a continuous lining enclosing the epiblasts by the so-called Rauber layer. In the third embryo, an epiblast cell, displaying clear signs of degeneration in the form of abundant swelling (presumptive necrosis), was protruding through a well-defined gap in the Rauber layer (Fig. 3).



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  		FIG. 3. a) Light micrograph of the inner cell mass of Day 9 bovine IVP embryo. Note the trophoblast cells (T1, T2, and T3) forming a complete covering (Rauber layer) on top of the inner cell mass consisting of hypoblast (H) and epiblast cells (E) of which some of the latter are apoptotic (AE) or necrotic (NE). b) Transmission electron micrograph of the same inner cell mass as in (a) displaying trophoblast (T) and hypoblast (H) cells as well as apoptotic (AE) and necrotic (NE) epiblast cells. c) Light micrograph of the inner cell mass of another Day 9 embryo displaying protrusion of a tentatively necrotic epiblast cell (NE) between two trophoblast cells (T1 and T2). Note the hypoblast (H) and the apoptotic epiblast cells (AE). d) Transmission electron micrograph of the same inner cell mass as in (c) displaying the necrotic epiblast (NE) protruding through a gap (G) between two trophoblast cells (T1 and T2)

All Day 14 embryos were elongated and displayed a well-defined mass of epiblast cells that, however, displayed even more abundant signs of apoptosis and necrosis than on Day 9. In all cases, the hypoblast cells had proliferated to form a continuous layer at the inside of the epiblasts as well as the trophoblasts. In three out of four embryos, degenerating (presumptive necrotic) epiblast cells protruded through gaps in the Rauber layer (Fig. 4).



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  		FIG. 4. a) Day 14 bovine embryo elongating within an agarose gel tunnel in the PHD system with defined inner cell mass (ICM) and elongating trophoblast (T). b) Embryo with invasive trophoblast (IT) in failures and cracks in the PHD gel. c) Light micrograph of a detail of a Day 14 IVP embryo. Note the necrotic epiblast cells (NE) protruding through a gap in the trophoblast (T1 and T2) as well as the underlying necrotic epiblast cells (NE) and the complete layer of hypoblast cells (H) in the embryo, which has collapsed during preparation

Gender Diagnosis

The percentage of males was higher (P < 0.001) in embryos of Quality I (97%, n = 38), but similar in Quality II (43%, n = 21) and Quality III (57%, n = 6) on Day 14.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In rodents and primates, post hatching culture was established several years ago [15–21] with rapid in vitro differentiation of in vivo–produced and flushed blastocysts. Recently, short publications have also reported post hatching development in bovine embryos produced entirely in vitro including fast growth, elongation [22–24], and signs of differentiation [26]. Our present work is, however, the first defining a stable in vitro culture system based on large material and documenting initial stages of differentiation, as well as revealing some of the factors important for such development.

According to our observations, in vitro post hatching developmental competence of bovine embryos is determined by several critical factors, including the hatching from the zona pellucida, morphology of embryos after hatching as well as the composition of media, and the three-dimensional structure applied to surround the cultured embryos.

Hatching is a demanding procedure for in vitro–produced bovine embryos, and a high percentage of embryos fail to hatch or rapidly degenerate after hatching. Supposedly, inadequate culture conditions or weakness of the embryo are main reasons for failure at this stage [36–39]. Although a detrimental effect simply from the contact of the embryo with the culture environment could also be supposed, earlier data prove that the zona pellucida is not essential for further development after compaction [40]. The embryo culture system used in our experiments was successful to support development of embryos without the zona pellucida even from the one-cell stage [41].

According to our data, the morphology of embryos after hatching determines their ability for further development. The differences among embryos are detectable with a stereomicroscope immediately after hatching. They increase during the course of development and can be used for qualification of embryos on Day 11, with predictive value regarding the further developmental competence.

Composition of the media used during post hatching development is a critical factor. Routinely, SOFaaci medium has proved to be adequate for embryo development [30], and it is presumed to be more appropriate in the continuous development. It is also known that the use of serum in culture has a positive effect on embryo development, although its effect in embryo culture medium is variable, depending on the serum origin, concentration, and embryo stage of exposure [40–42]. In contrast to the earlier developmental stages, fetal bovine serum supplementation had a superior effect on post hatching embryo survival and development compared with cattle serum [25, 26]. Other studies have revealed that the energy demands of bovine embryos increases substantially after compaction, blastocoele formation, and expansion, enhancing glycolytic activity [43] and glucose uptake [44]. Accordingly, supplementation of the medium with high concentrations of glucose considerably increased the developmental ability of embryos after hatching (data not shown), and glucose was therefore used as a standard component of the PHD medium. However, at the present stage we do not state that glucose is the ideal source, because other energy substrates need to be tested in the running system. So far, we can only affirm that glucose was able to support embryo growth and elongation in a high rate.

Considering the high metabolic level of the embryo, elevated production and accumulation of detrimental factors such as ammonia [45] and oxygen-derived radicals in the culture medium might occur [46]. This could suggest a positive effect of renewing the culture medium. However, regular medium changes in the PHD system had no positive effect on embryo development (data not shown). Furthermore, the simple act of removing the PHD dishes from the incubator on Day 12 negatively influenced embryo elongation and increased degeneration. Changes in temperature, pH, or the microenvironment of the embryos might participate in this phenomenon.

Based on earlier observations [23], we applied a three-dimensional gel structure to induce elongation in vitro. In vivo, the elongation is initiated in the uterus around Day 12 [29–31] and is fundamental to the interaction with the endometrium, maternal recognition, and normal development [47]. It may also play an important role in formation and orientation of the embryonic disc. So far, there is no direct evidence regarding the essential role of the three-dimensional structure of the uterus in inducing and orienting elongation. In our study, eventually the embryos floated out of the agar tunnels because of irregular inclination of the tunnel or oversized embryos, and in those cases the elongation never occurred spontaneously. These facts suggest the need of the tubular structure in the elongation process and reinforce the hypothesis that a similar mechanism might occur in vivo. In our system the tunnels were used, but other structures might be useful too.

According to our observations, high serum supplementation of the agar gel positively influenced the initiation of elongation, showing that embryos are able to benefit from components or factors included in its surrounding. Whether the serum in the gel was used as nutrient source or simply as physical positive component in the gel structure is not known. Our finding that the trophoblast follows and invades ruptures or depressions in the gel surface indicated a need for mechanical contact with the surface and a positive effect of higher serum concentration in the gel. It could also indicate that both nutritional and physical benefits were present in the gel.

The established system was suitable to induce elongation and initial steps of differentiation of the embryonic disc. The mean length of embryos elongating in the PHD system ranged from 1.3 to 7.6 mm on Day 14 and 1.8 to 7.8 mm on Day 15, and it was very dependent on the embryo quality. In in vivo–produced embryos, a range of 0.5–19.0 mm was reported on Day 14 [23]. Bertolini [30] found a range of 1.1–282.0 mm for in vivo–produced and 1.8–122.0 mm for in vitro–produced embryos on Day 16. Thus, the variation of the length seems to be characteristic for embryos developing either in vivo or in vitro after hatching. Generally, the level of elongation in the PHD system was compromised compared with that seen in vivo [10], probably as the result of the suboptimal environment that our system could provide. However, according to our knowledge this system is the first that induces in vitro elongation in a relatively high proportion of bovine embryos produced entirely in vitro.

An important improvement of the PHD system was the identification of differentiated cells already on Day 9 embryos, with an abundant penetration of epiblasts through the Rauber layer on Day 14. In vivo, the first sign of differentiation is found by Day 8, when the inner cell mass delaminates hypoblast cells [31]. Around Day 10, the hypoblast cells form a confluent layer lining at the inside of the trophoblast. By Day 12, the inner cell mass is defined as an epiblast and is overlaid by a thin layer of trophoblast referred to as the Rauber layer [48], establishing the embryonic disc. In the present study, the identification of the hypoblast and epiblastic cells, with the definition of the Rauber layer, indicates that normal steps in the embryonic development have occurred in the PHD system. However, the progressive degeneration observed in the epiblastic cells suggests that the culture system still needs further improvement. On the other hand, the presence of a degenerative process already on Day 9 embryos points out that the period just after hatching is critical for the normal in vitro development of differentiated cells. Therefore, that period should be the focus in future studies and development of optimized culture systems.

Gender determination of our elongated embryos showed in an overwhelming majority of male embryos. A similar, but much less prominent, imbalance in favor of males among in vitro–produced bovine blastocysts has been reported earlier by some [49–53] but not all publications [30]. The difference in sex ratio has been attributed to several factors such as gene expression [50, 54]; detoxification of oxygen radicals [54]; developmental speed [49, 52]; oocyte maturation and fertilization [51, 52, 55]; and laboratory conditions [55]. Moreover, Gutierrez-Ádan [52] found that higher glucose concentration in embryo culture medium is more detrimental to female embryos.

Several questions remain to be investigated related to the post hatching culture of bovine embryos. Nevertheless, the innovative information produced with the PHD system increases the expectation of its future uses for monitoring in routine systems based on embryo development in vitro, especially for cloned embryos; to contribute in basic biological studies; or to investigate toxicity of in vitro culture products, pharmaceuticals, pesticides, and other chemicals. An additional potential application for this technique is to become a potential source of totipotent and pluripotent cells for the study of stem cells, considering that the PHD system allows the maintenance of the embryo during the ideal period for such work.

In conclusion, our investigation is the first report of the in vitro culture of post hatching bovine embryos based on a large material and with a defined and stable system. The PHD system has proven to be capable in promoting fast growth, elongation, and initial signs of differentiation, documenting its perspective as a novel and useful tool for embryo evaluation.


   ACKNOWLEDGMENTS
 
Professor Gabor Vajta is thanked for sharing his knowledge, experiences, and ideas in this new field. Anette Pedersen, Ruth Kristensen, Klaus Villemoes Gunnel, and Inge Lise Sørensen are thanked for expert technical assistance. Dr. Margot Dode is thanked warmly for excellent support in the manuscript preparation.


   FOOTNOTES
 
1 Correspondence: FAX: 55 61 3403658; dobrandao{at}uol.com.br Back

Received: 15 December 2003.

First decision: 1 January 2004.

Accepted: 7 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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