Embryo handling and evaluation

Identification and evaluation of embryos is one of the most challenging aspects that confronts the embryo transfer practitioner, especially the beginner. Embryo quality and poor handling techniques can directly affect pregnancy rates. A stepwise procedure for embryo searching is presented at the end of this section.

ENVIRONMENT

Once removed from the stable, protective environment of the uterus the embryo should be handled with respect regarding its temperature, pH, osmolarity and contaminants-factors which may affect viability.

Embryos may be maintained at room temperature for several hours without decreasing pregnancy rates significantly, provided the embryos are transferred to fresh holding medium every two hours. Storing embryos in an ice-water bath is recommended for long-distance transportation. There is no obvious decrease in pregnancy rates after storage at this temperature for 12–24 hours. Embryos do not tolerate temperatures above body temperature (39°C) very well.

The physiological range of proper pH for embryos is from 7.1 to 7.5. Thus, the pH of flushing and holding media needs to be within this range. Phosphate buffered saline (PBS) is commonly used because under atmospheric conditions its pH changes very little. However, if bicarbonate (NaHCO₃) buffered media (Ham's F-10, MEM, TCM-199, etc.) are used, care must be taken to avoid large shifts in pH (rising pH) when the embryo is exposed to an atmosphere high in CO₂.

Even a slight change in the salt concentration (osmolarity) of the medium can effectively reduce the viability of embryos. If the salt concentration of the flushing or holding media is below that of the uterine environment, embryos will absorb water and swell to reach osmotic equilibrium, which sometimes results in rupture of the cell membrance. Conversely, if the salt concentration is above that of the uterus, the embryo will shrink in size (dehydration) causing a reduction in metabolic activity. Comparing the two situations, although both are deterimental to embryos, shrinkage would be less detrimental. If PBS is prepared from a powdered mixture, care should be taken that the correct amount of water is added. The normal osmolarity of uterine fluid is 270–300 mosm.

Exposing the embryos to ultraviolet rays for a prolonged period may cause cellular death. The use of insecticide sprays in the embryology room should be avoided. Insufficient time of aeration after using ethylene oxide gas for sterilization of equipment is detrimental to live cells. Storage period, different suppliers and batches (lot number) of sera all affect embryo growth differently.

IDENTIFICATION OF EMBRYOS

Identification of the embryo in the uterine effluent is based on several morphological features of the embryo, which is the only practical method to determine suitability for transfer. Such methods are subjective and depend very much on experience.

The embryo is spherical and is composed of cells (blastomeres) surrounded by a gelatin-like shell, an acellular matrix known as the zona pellucida. The zona pellucida performs a variety of functions until the embryo hatches, and is a good landmark for embryo identification. The zona is spherical and translucent, and thus is clearly distinguishable from cellular debris. Because of its shape the embryo tends to roll on the bottom of the (searching) dish.

Diagrams of normal bubaline embryos at various stages of development are presented in Figure 4. The stage of development after fertilization must be appropriate for the day on which the embryo is collected, but is not precisely known for buffalo embryos since only relatively few have been recovered to date, with most of them recovered on day 5 to day 7 after the onset of oestrus. The rate of development of water buffalo embryos is faster than that of cattle embryos (Drost and Elsden, 1985). Development from the morula stage to escape from the zona pellucida (hatching) appears to occur in one day in buffaloes. Morphologically water buffalo embryos are similar to bovine embryos and general morphological descriptions can be adapted.

The overall diameter of the bovine embryo has been estimated to be 150–180 μ including a zona pellucida thickness of 12–15 μ. The diameter remains constant until expansion of the blastocoele begins. The colour of the morula and (early) blastocyst also facilitates identification because the embryo is usually darker than other uterine debris. Knowing the age of the embryo (days post oestrus) is also very important in locating the embryo in the searching dish. The fully expanded blastocyst possesses a thinner zona pellucida and is pale (translucent) in colour. A spontaneously hatched embryo is very hard to identify because the embryonic mass, without the zona pellucida, is morphologically similar to uterine debris. If the hatched embryo has collapsed, it may still be identifiable but with considerable difficulty. In summary, the important criteria in identifying embryos are: (1) shape of the embryo; (2) presence of a zona pellucida; (3) size; (4) colour; and (5) knowledge of the age of the embryo.

During embryonic development, cell numbers increase geometrically (1- 2-4-8-16- etc.). Synchronous cell division is generally maintained to the 16-cell stage in embryos. After that, cell division becomes asynchronous and finally individual cells possess their own cell cycle. The cells composing the embryos are termed blastomeres and are easily identified by the 16-cell stages as spherical cells. After the 32-cell stage (morula stage), embryos undergo compaction. As a result, individual cells in the embryo are difficult to identify beyond this stage. The most obvious morphological manifestation during compaction is the loss of a concise cellular outline. The outline of the cells becomes smooth as the cells flatten against one another to maximize their surface contact. It has been shown that both intracellular and intercellular activity in mammalian embryos before and after compaction are very different. At the intercellular level, specialized junctions form between individual blastomeres. These specialized junctions are called gap junctions or focal tight junctions. The tight junctions form an interlocking network providing a permeable seal between the core of the embryo and the external environment. At the intracellular level, polarity develops within individual cells, and microvilli become restricted to the outer surface of the cell. The dynamic cell differentiation begins during the formation of the blastocyst after compaction is completed. Two distinct cell types are present at the blastocyst stage. The two types of cells differ in their morphology, biochemistry, developmental potential and eventual expression. The embryo proper develops from the inner cell mass whereas the surrounding trophectoderm primarily gives rise to the chorionic ectoderm of the placenta. The mechanism that underlines this cellular differentiation is still not well understood.

FIGURE 4
Diagrams of normal, excellent quality embryos at different stages of development

HANDLING THE EMBRYOS

Once an embryo is identified in the searching dish, it is immediately transferred to a small Petri dish (35 × 10 mm) containing fresh, filtered (0.22–0.45 μ pore size), sterile medium. As a holding medium, generally phosphate buffered saline (PBS) containing penicillin plus 10–20 percent heat inactivated serum is used. Embryos are tentatively classified simply as good or bad, and may be recorded on the cover of the holding dish. This allows for a quick estimate of the total number of embryos found. Embryos are then serially rinsed through at least three different dishes containing fresh sterile medium using a new sterile pipette for each step. Finally, they are placed in a dish awaiting transfer or cryopreservation. Under certain circumstances, e.g. for export, embryos must be rinsed through ten different dishes containing sterile media. All dishes must be kept covered between searches to avoid contamination, and particularly evaporation, when placed in the incubator. Evaporation of the small volume of medium in a flat dish rapidly leads to hypertonic solutions.

CLASSIFICATION OF EMBRYOS

Embryos recovered five to seven days after oestrus are classified morphologically into the following groups (Figure 4).

Morula

Blastomeres are round in shape and are not tightly connected to each other. Individual blastomeres are difficult to discern from one another. The cellular mass of the embryo occupies most of the perivitelline space.

Compact morula (tight morula)

The shape of a tight morula is similar to a golf ball, in that the outer edge is slightly bumpy (scalloped) in appearance because of compaction. Individual blastomeres are no longer distinguishable. Cells on the surface of the mass are polygonal in shape. The embryo mass occupies 60–70 percent of the perivitelline space.

Early blastocyst

A tiny clear space is visible which contains fluid. This area is the beginning of the blastocoele. The embryo occupies 70–80 percent of the perivitelline space.

Blastocyst

The prominent blastocoele cavity comprises more than 70 percent of the volume of the embryo. Two groups of cells are present and clearly recognizable as the trophoblastic layer beneath the zona pellucida and the darker inner cell mass occupying one side of the embryo. The perivitelline space may still be visible.

Expanding or expanded blastocyst

There is no perivitelline space between the layer of trophoblastic cells and the inside of the zona. The zona pellucida becomes thinner as the blastocyst expands. A small (well compacted) inner cell mass positioned on one side of the embryo is observed. The colour of the embryo is pale to clear because of the large amount of fluid present inside.

Hatched blastocyst

Ultimately the blastocyst expands to the point of rupture and the embryo escapes from the disrupted zona. Hatched blastocysts may be spherical with a well-defined blastocoele or they may be collapsed, resembling debris. Identification of embryos at this stage can be difficult for the inexperienced operator. When zona-free, or hatched, blastocysts are collected, there is a greater risk of damage due to handling. Furthermore, hatched blastocysts are “sticky” and may adhere to tubing and glassware. Embryo filters should not be used when there is a possibility that hatched embryos will be recovered (> day 7).

FIGURE 5
Diagrams of good quality embryos

Embryos are generally classified into different groups based on gross morphological appearance (Figures 5, 6, 7).

Excellent embryos have no visible imperfections.

Good embryos have a few recognizable imperfections, such as poor compaction, variation in cell size or a few extruded cells.

FIGURE 6
Diagrams of fair quality embryos

Fair embryos show more disarrangement, such as a small embryonic mass with irregular shape, and large numbers of extruded or dead cells. They are usually one to two days retarded in development.

Poor embryos show signs of cellular degeneration, a very small embryonic mass and many extruded cells or disintegrated cytoplasm. Generally, development is retarded by more than two days in these embryos.

Very poor embryos contain either all dead cells or only a few live cells or a very tiny cell mass which is extremely disorganized in appearance. They are composed mainly of debris and generally are not worth transferring.

FIGURE 7
Diagrams of poor and very poor quality embryos

Additional criteria may be considered for evaluation of embryos to provide a fair assessment:

• Age of embryo from oestrus to collection.
• Number of cells present in the embryo.
• Compactness of the blastomeres. Loosely arranged blastomeres may indicate underdevelopment.
• Cellular mass of the embryo should be spherical and symmetrical.
• Colour of the embryo. A very dark colour, often seen in damaged frozen embryos, may indicate lipid accumulation inside the cells.
• Variation in cell size. Healthy embryos ideally have homogeneous blastomeres of the same size.
• Vesicles in the cells. Although the physiological roles and properties of vesicles seen in cells have not been well described in the literature, embryos with large vesicles tend to develop poorly in vitro.
• Number of blastomeres extruded from the main cell mass. Extruded cells in a compacted morula or blastocyst are easily identified in the perivitelline space. Once the blastocyst has expanded, filling the entire perivitelline space, identification of the extruded cells is very difficult. It is believed that extruded cells are not incorporated into the main cell mass during compaction. Usually, a few small extruded cells will not interfere with further embryonic development. A large number of small extruded cells or the presence of many dead cells will hinder proper embryonic development.
• Percentage of perivitelline space occupied by the main cell mass is important in evaluating the cell mass size.
• Knowledge of the age of the embryo is also necessary to assess the proper stage of development according to the day on which it is collected.

There are additional methods for the evaluation of the viability of embryos:

• Culturing in vitro. If embryos are of doubtful quality (transferable or not), observing further development in vitro is helpful.
• Culturing in vivo. Temporary transfer to the ligated oviduct of rabbits or sheep and re-recovery to determine further development.
• Staining cells. Identification of both live and dead cells under the fluorescent microscope can be made through incubation of embryos with fluorescent dyes, such as fluorescein-diacetate or diamidine-2-phenylindole (Hoppe and Bavister, 1983/84; Schilling et al., 1979).
• Measuring metabolic activity. The rate of glucose incorporation into the embryo during culture has been examined.
• Transferring into recipients. The best test is the pregnancy rate after transfer. However, many factors including the transfer method, synchrony and skill of the operator affect pregnancy rates.

TABLE 4
Effect of embryo quality on the pregnancy rate in cattle

* Percentage of pregnancies
** Number of embryos transferred

PROCEDURE FOR EMBRYO SEARCHING

1. Embryo searching equipment is prepared before the uterus of the donor is flushed when a concentrating filter (Figure 8A) is used.
2. One to three 100 × 100mm square, grid-bottom dishes are used to search one filter (Figure 8B).
3. Each dish is labelled with the donor's number and the sequence in which it will be filled from the filter.
4. Fresh PBS is drawn into a 30- or 35-ml syringe with sterile precautions. A 22-gauge 1-inch needle is then attached to the syringe. This syringe is used to rinse the filter. The presence of serum in the PBS must be avoided during this procedure to prevent foaming.
5. The medium, left in the filter from the flush, is swirled and poured into dish 1.
6. The filter is held at an angle and rinsed into the dish. More fluid may be needed if the filter contains mucus. Steps 4 and 5 are repeated using as many dishes as necessary, until the filter is completely clear (no mucus and/or tissue debris left on the filter). Each dish will safely hold 75 ml.
7. 5–7 ml of serum are added to the searching dish and stirred in the medium while clearing bubbles from the edge of the dish.
8. Dishes are searched under a stereomicroscope (15X). The dish is systematically moved along the reference lines to ensure the entire dish is searched (Figure 9). When embryos are identified, they are immediately transferred into a small Petri dish (35 × 10mm; Figure 8C) containing sterile, filtered holding medium (PBS + 10–20 percent serum). All dishes should be kept covered between searches to avoid contamination, and particularly evaporation, when placed in the incubator.
9. The embryo is drawn into a micropipette attached to a 0.5-ml syringe (Figure 8E) from the searching dish while observing under the microscope. Then, the embryo is transferred to the small holding dish. The number of good and/or bad embryos is tentatively recorded on the lid of the holding dish. The estimated number of corpora lutea usually serves as a guide to the number of embryos to be searched for.
10. After all the embryos from one donor are accumulated in one small holding dish, they are transferred three times, each time to a dish containing freshly filtered PBS plus serum. This procedure serves to rinse the embryos.
11. The embryos are now graded and prepared for the next procedure: culturing, transferring, freezing, splitting or sexing.
12. Grading is facilitated under higher magnification (100X) with a regular light microscope, preferably an inverted microscope with adequate working room on the stage.

FIGURE 8
Equipment for embryo searching. A. Embryo filter; B. Searching dish with grid; C. Holding dish; D. 35-ml non-toxic syringe with microfilter attached; E. 0.5-ml syringe with catheter tip.

Alternatively, when a large 500–1 000-ml graduated cylinder is used, the embryos are allowed to settle to the bottom of the cylinder for 20–30 minutes. All but the bottom 75 ml of flushing medium is slowly siphoned off with a small diameter piece of tubing. The final 75 ml of medium is gently swirled and then poured into a searching dish. The cylinder is rinsed two to three times with small volumes of flushing medium (containing 1-percent serum) and emptied into a searching dish. This is followed by steps 7 to 12 as before.

LOADING THE STRAW

Just prior to non-surgical transfer embryos are loaded individually in sterile 0.25-ml French straws. The embryo is aspirated from the holding dish into the straw with the aid of a 1-ml tuberculin syringe attached to the plug end of the straw. First a 3-cm column of medium is aspirated, followed by a 0.5-cm column of air, then a 3-cm column of medium containing the embryo, followed by another air bubble. The remainder of the straw is filled with medium until the initial column of medium wets and solidifies the plug (Figure 10C).

FIGURE 9
Searching grid-bottomed dish under stereomicroscope.

FIGURE 10
Equipment for non-surgical embryo transfer. A. Miniaturized embryo transfer syringe, length 52 cm, with sheath and covered by a sanitary, plastic sleeve; B. Standard Al syringe, length 44 cm, with sheath and covered by a sanitary, plastic sleeve; C. 0.25-ml straw loaded with an embryo in the centre column of medium flanked by two air bubbles.