Evaluation of In Vivo-Derived Bovine Embryos

Chapter 79
Evaluation of In Vivo-Derived Bovine Embryos

Marianna M. Jahnke1, James K. West2 and Curtis R. Youngs3

1 Veterinary Diagnostic and Production Animal Medicine Department, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA

2 Director of Embryo Transfer Services, Iowa State University, Ames, Iowa, USA

3 Department of Animal Science, Iowa State University, Ames, Iowa, USA


Morphological evaluation of embryo quality is performed for three major reasons. First reason is to differentiate between embryos and unfertilized ova. Careful evaluation of harvested embryos/ova is necessary to ensure that viable embryos are not discarded or, conversely, that unfertilized ova are not transferred or cryopreserved. The second reason to perform morphological assessment of embryos harvested from a donor is to determine if the developmental stage of the embryos is consistent with the expected developmental stage based on the days since estrus that the embryos were collected. There is often a high degree of variability in observed stage of embryonic development among embryos obtained during embryo recovery from a single donor female, and this variation in stage of development should be considered when selecting a suitable recipient female. The third reason to assess embryo quality is to enable technicians to have sufficient information on which to base the decision to transfer or to cryopreserve the harvested embryos. Poorer-quality embryos that may result in pregnancies if transferred fresh usually do not survive the cryopreservation process.

Despite the importance of this topic, embryo evaluation is a difficult aspect of the overall embryo transfer process to learn, especially if attempting to self-train. Although this chapter will provide numerous helpful photographs of embryos and unfertilized ova, beginners would benefit from joining an experienced embryo transfer mentor for intensive and repetitive training sessions on morphological embryo evaluation. Proper training, proper equipment, and extensive experience are the three major factors affecting one’s proficiency with embryo evaluation.

History of bovine embryo evaluation

The very earliest stages of bovine embryonic development (ovum and 2-cell embryo) were first described in 1931 by Hartman et al.1 In the following decade two other research groups2,3 described and illustrated various stages of bovine embryonic development. These very early descriptions of embryonic development served as the foundation of the morphological embryo evaluation method that subsequently evolved and became widely used in the commercial bovine embryo transfer industry.4

The morphological embryo evaluation method depends on the human eye to critically evaluate subtle differences among harvested embryos. During the early years of its use, the method was criticized as being too subjective, inconsistent, and prone to error. As a result, several alternative methods for assessing embryo quality were investigated for their potential to be less subjective and more consistent than morphological evaluation. Some of these methods included the ability of an embryo to continue its development during in vitro culture,5,6 dye exclusion test,7 measurement of embryonic metabolic activity,6 glucose uptake,8 live–dead stains,9 computerized image analysis,10 fluorescein diacetate vital stain,11,12 DAPI (4′,6′-diamidino-2-phenyl-indole) staining for dead cells,13 and in vivo culture in rabbit oviducts.14

Refinement of the morphological embryo evaluation method

Despite the numerous attempts to develop a more precise and predictable method to evaluate the viability of bovine embryos, no methods have been devised that are considered better or more accurate than morphological embryo evaluation. This method is the preferred method of embryo viability assessment under clinical conditions because it is a relatively simple and fairly rapid technique for evaluation of bovine embryos.

After the early report on bovine embryo evaluation in 1976 by Shea et al.,4 three other landmark papers on embryo evaluation were published within the next 7 years.15–17 These two groups of practitioners and scientists described the criteria that are, for the most part, still used today as a general guideline for embryo evaluation and prediction of potential pregnancy. The latest of those papers17 described numerous parameters commonly used for embryo evaluation, including stage of embryonic development and structural features such as embryo size and shape, presence of extruded cells or degenerated cells, color characteristic, number and compactness of the cells (also called blastomeres) that comprise the early embryo, integrity of the zona pellucida, and presence or absence of vacuoles within the cytoplasm of the embryonic blastomeres.

History of the IETS grading and classification system

The International Embryo Transfer Society (IETS) is an international organization that was born in 1974 in Denver, Colorado, USA. It was created by a group of North American practitioners and scientists who recognized the need to share scientific discoveries. As the worldwide commercial embryo transfer industry grew, it became critical to develop standardized embryo nomenclature so that buyers and sellers of embryos knew precisely the stage and quality of embryos being marketed.

The IETS published in their manual18 a recommended two-digit coding system to uniformly and systematically describe embryos and their characteristics. On completion of the morphological assessment of an embryo, it is assigned two numbers, separated by a hyphen, that correspond to the embryonic stage of development and embryo quality grade. The standardized coding system used to describe the stage of embryonic development ranges from 1 (an unfertilized oocyte) to 9 (expanding hatched blastocyst). The standardized code for embryo quality, based on morphological characteristics of embryos, is also numerical and ranges from 1 (excellent/good) to 4 (dead/degenerating). This IETS embryo grading and classification system must be used for international embryo trade, and because of this mandate most practitioners have adopted this system for everyday use. A more detailed description of the IETS embryo grading and classification system is discussed later in this chapter.

The importance of a good microscope

Before delving into the specific methodology used for morphological evaluation of embryos, it is important to remind readers that proper equipment is needed for accurate embryo evaluation. Although many beginners in embryo transfer are tempted to purchase an inexpensive microscope as a means to reduce start-up costs, doing so will likely create difficulties in finding and evaluating embryos. A poor-quality microscope can make it extremely challenging for a practitioner to tell the difference between an unfertilized ovum (UFO) and a compact morula. Similarly, a practitioner will be hampered by a poor-quality microscope when attempting to discern the various embryo quality grades.

Unlike semen evaluation, where a compound microscope is used, embryo transfer requires the use of a stereomicroscope. Stereomicroscopes, also called dissecting microscopes, are designed for use with three-dimensional specimens. Preimplantation bovine embryos, although microscopic in size, are round like a basketball and thus are best evaluated using a stereomicroscope.

There are several important features of a stereomicroscope that can directly influence one’s ability to easily and accurately evaluate embryos. One important feature is the overall magnification. Overall magnification of a stereomicroscope is a function of the magnification of the eyepieces, objective lens, and zoom changer. For example, total magnification of a stereomicroscope equipped with 10× eyepieces, a 1× objective lens, and a 2× zoom changer would be approximately 20× (10 × 1 × 2 = 20). Many technicians prefer to search for embryos at a total magnification between 10× and 15×, but to effectively evaluate the fine details of an embryo and to identify any abnormalities, such as cracks in the zona pellucida, embryos should be observed under a good optical quality stereomicroscope with a magnification range that reaches at least 50×. It should be noted that the IETS-approved procedure for sanitary handling of embryos requires that embryos be inspected at a magnification of at least 50 × .

Most embryo transfer technicians prefer that the eyepieces not exceed 10× in order to maintain an adequate field of view and image resolution. A microscope equipped with a diopter adjustment on one eyepiece (if not both) is very useful for compensating differences in eyesight between a technician’s left and right eyes. An adjustable binocular tube will also enable multiple technicians with varying interpupillary distances to use the same stereomicroscope.

A second important feature of a stereomicroscope for embryo handling and evaluation is the working distance (distance from the embryo to the objective lens). The working distance should be long to give maximal flexibility when searching for embryos and the microscope stage should be large and free of obstacles such as stage clips, power buttons, or similar items that make sliding of Petri dishes on the stage difficult. A large and clear stage is important for providing adequate space to move Petri dishes and to operate the embryo handling device without accidently spilling the Petri dish and losing the embryos.

A third important feature of a stereomicroscope used for embryo evaluation is the illumination. The illumination should come from underneath the stage plate and it should be bright and uniform across the entire stage plate. A clear stage plate is needed; frosted stage plates are not suitable for embryo work. Brightfield illumination is standard for most stereomicroscopes, but darkfield illumination can be helpful when searching through “cloudy” fluids in a search dish.

Normal in vivo bovine embryonic development

In order to know whether embryos harvested from a donor have developed at the expected rate, it is important for technicians to have a good understanding of normal in vivo bovine embryonic development. The overall diameter of a bovine embryo is 150–190 μm (or about 15 times the size of white blood cells), which includes the zona pellucida thickness of approximately 12–15 μm.17 The overall diameter of the zona pellucida-enclosed embryo remains virtually unchanged from the 1-cell stage of development until the blastocyst stage of development because blastomeres (individual cells of a fertilized embryo prior to cellular differentiation at the blastocyst stage of development) become progressively smaller with each cell division.

The zona pellucida is a translucent shell surrounding an embryo from the 1-cell until the expanded blastocyst stage of development. The zona pellucida functions as a physical barrier against pathogens, contains receptors for sperm, and blocks accessory sperm from reaching the ovum. Once an embryo reaches the expanded blastocyst and continues to grow, the embryo becomes so large that it ruptures the zona pellucida and “hatches.”

During normal in vivo embryonic development, blastomeres go through a series of cleavage divisions. The ovum, after fertilization, divides and develops into a 2-cell embryo, the 2-cell develops into a 4-cell, the 4-cell into an 8-cell, and so on. When the blastomeres appear like a cluster of grapes and individual blastomeres cannot be differentiated, this stage of embryonic development is known as the morula stage. As the embryo further develops, it prepares to undergo its first differentiation event known as blastulation. Just prior to this differentiation event, cells of the morula “compact” and are allocated to the “inside” and “outside” parts of the embryo.

Once blastulation has occurred, the embryo is at the blastocyst stage of embryonic development. The blastocyst contains two populations of differentiated cells: an outer ring of trophoblast cells and an inner clump of cells known as the inner cell mass. The inner cell mass (ICM) will give rise to the fetus and most layers of the placenta, while the trophoblast cells will eventually form the outermost layer of the placenta. The blastocyst stage of embryonic development has another distinguishing feature, the presence of a blastocoele cavity. This fluid-filled cavity is surrounded by the trophoblast cells and grows progressively larger to help cause thinning of the zona pellucida prior to embryo hatching. Figure 79.1 illustrates the features observed in a blastocyst-stage bovine embryo, while Figure 79.2 shows the stages of bovine embryonic development.


Figure 79.1 Illustration of a blastocyst-stage bovine embryo.


Figure 79.2 Normal embryonic development of bovine embryos.

Reproduced with permission from Stringfellow D, Givens M. Manual of the International Embryo Transfer Society, 4th edn. Champaign, IL: International Embryo Transfer Society, 2010.

IETS grading and classification system

Embryos are typically recovered 6–8 days after the onset of estrus. Harvested embryos should be classified into groups based on their stage of embryonic development and quality grade. Table 79.1 illustrates the current IETS embryo evaluation system followed by a detailed description of these developmental stages. After a technician completes the embryo evaluation process, a two-digit code is assigned to each embryo. For example, a fair-quality early blastocyst would be denoted as 5-2. A blastocyst-stage embryo of excellent or good quality would be denoted as 6-1.

Table 79.1 IETS recommended codes for embryo stage of development and embryo quality grade.

Source: Stringfellow D, Givens M. Manual of the International Embryo Transfer Society, 4th edn. Champaign, IL: International Embryo Transfer Society, 2010.

Stage of development

  1. Unfertilized
  2. 2-cell to 12-cell
  3. Early morula
  4. Morula
  5. Early blastocyst
  6. Blastocyst
  7. Expanded blastocyst
  8. Hatched blastocyst
  9. Expanded hatched blastocyst
Quality of embryos

  1. Excellent or good
  2. Fair
  3. Poor
  4. Dead or degenerating

Stage of embryonic development

Stage code 1: 1-cell

For any entity recovered on days 6–8 that is truly only a single cell, it is safe to assume that it is a UFO. However, a challenging task for an inexperienced technician is to distinguish a UFO from a compact morula (stage code 4, see below). Discerning a UFO from other stages of embryonic development can also be difficult because not all UFOs have the same morphological appearance. Many “normal” UFOs have a perfectly spherical zona pellucida, perfectly spherical oolemma (also called the vitelline membrane), relatively uniform granularity in the cytoplasm, and a moderate volume of perivitelline space. However, other UFOs may show varying signs of degeneration including cytoplasm fragmentation, which can lead to the illusion of a blastocoele cavity and/or appearance of cell division, and extreme condensation, which can lead to confusion with a compact morula.

Stage code 2: 2-cell to 12-cell

Cells of a preimplantation embryo prior to morphological and functional differentiation are called blastomeres. Any embryo recovered 6–8 days after the onset of estrus that contains 2–12 blastomeres is almost certainly dead or degenerate. Under normal conditions this stage of embryonic development is expected to be present in the oviduct (not the uterus) prior to day 5 of the bovine estrous cycle (day 0 = onset of estrus). Clearly, if a 2- to 12-cell embryo were somehow still viable, its development is severely delayed and it should not be considered for transfer or cryopreservation.

Stage code 3: early morula

The term “morula” has its origin in the Latin word for mulberry. Embryos at the morula stage of embryonic development consist of a group of at least 16 cells. Although some individual blastomeres are easy to visualize, others are only partially visible as they are “hidden” by other blastomeres. This stage of embryonic development, like all stages of development discussed previously, is not well suited for cryopreservation.

Stage code 4: compact morula

Individual blastomeres present in this embryo have coalesced to form a tightly compacted ball of cells. It is impossible to discern individual blastomeres at this stage of embryonic development. As a part of the compaction process, cells are allocated to the “inside” part of the ball of cells and also to the “outside” part of the ball of cells. The cells on the outside of the ball form tight junctions with one another in a contiguous ring. The cells comprising the embryo occupy approximately 60–70% of the space inside the zona pellucida (called perivitelline space). As stated earlier, it is this stage of embryonic development that can be easily confused with a UFO.

Stage code 5: early blastocyst

The most distinguishing characteristic of an early blastocyst-stage embryo is the presence of a small fluid-filled cavity known as the blastocoele. Several differentiated trophoblast cells will also be visible between the blastocoele cavity and the zona pellucida, although the ability to distinguish them from the cells of the ICM is quite limited at this stage. The cells comprising the embryo occupy approximately 70–80% of the perivitelline space. The zona pellucida is still the same thickness as the less developmentally advanced stages of embryonic development previously discussed.

Stage code 6: blastocyst

A blastocyst-stage embryo will have a clearly defined trophoblast layer, blastocoele cavity, and group of ICM cells, along with a normal-thickness zona pellucida. There will be little to no perivitelline space present except in cases where the embryo may be partially collapsed.

Stage code 7: expanded blastocyst

The most distinguishing feature of an expanded blastocyst-stage embryo is an increase in its overall diameter, 1.2–1.5 times larger than its original diameter of 150–190 μm, coupled with a thinning of the zona pellucida to approximately one-third of its original thickness of 12–15 μm. Because of the large increase in the volume of the blastocoele cavity at this stage of embryonic development, no perivitelline space is present. Interestingly, expanded blastocyst-stage embryos frequently appear collapsed. This phenomenon is characterized by a partial loss of blastocoele cavity fluid and is believed to be a normal phenomenon that contributes to embryo hatching. The zona pellucida, once thinned, never regains its original thickness.

Stage code 8: hatched blastocyst

Hatched blastocyst-stage embryos can be undergoing the process of hatching or may have completely hatched from the zona pellucida. Embryos at this stage may be spherical with a well-defined blastocele cavity or may be collapsed. Identification of this stage of embryonic development can be quite difficult if the embryo has completely hatched from the zona pellucida and has nearly totally collapsed because it can easily be mistaken for a piece of endometrial tissue. This stage of embryonic development is not eligible for international trade because the nonintact zona pellucida makes it impossible to wash the embryo in accordance with IETS procedures for sanitary handling of embryos.19

Stage code 9: expanded hatched blastocyst

This stage of embryonic development is identical in appearance to a completely hatched stage code 8, except for being significantly larger in diameter. It is uncommon to recover expanded hatched blastocysts from bovine donor females unless flushing is performing more than 8 days after the onset of estrus. This stage of embryonic development is not eligible for international trade.

Embryo quality grade

Embryo quality grade is determined by visual assessment of an embryo’s morphological characteristics. Characteristics used to determine the quality grade of an individual embryo include uniformity of blastomeres (size, color, and shape), presence of extruded cells, presence of dead/degenerating blastomeres, degree of cytoplasmic granularity, presence of cytoplasmic vacuoles, cytoplasmic fragmentation, and structural integrity of the zona pellucida.

Quality grade code 1: excellent or good

A quality grade 1 embryo has a symmetrical and spherical embryo mass with individual blastomeres that are uniform in size, color, and density. The embryonic stage of development is consistent with that expected based on the day of the donor female’s estrous cycle when the embryo was recovered. Irregularities (such as cytoplasmic vacuoles), if any, are relativity minor, and at least 85% of the embryonic cells are an intact, viable and cohesive mass (i.e., no more than 15% of embryonic cells are extruded into the perivitelline space). The zona pellucida should be spherically shaped, smooth, and with no concave or flat surfaces that might cause the embryo to adhere to a Petri dish or straw.

Quality grade code 2: fair

Moderate irregularities exist in the overall shape of the embryonic mass or in the size, color and density of individual cells comprising the embryo. Mild unevenness of cytoplasmic pigmentation within individual blastomeres (do not confuse this with translucency of the blastocoele cavity and darkness of the ICM cells of a blastocyst-stage embryo), “peppering” of cytoplasm with individual blastomeres, and/or a small number of vacuoles within the cytoplasm of blastomeres would be sufficient to warrant a quality grade 2 score. At least 50% of the embryonic cells should be an intact, viable and cohesive embryonic mass.

Quality grade code 3: poor

Major irregularities exist in the shape of the embryonic mass or in the size, color and density of individual cells. Extreme unevenness of cytoplasmic pigmentation within individual blastomeres, significant “peppering” of cytoplasm within individual blastomeres, and/or large numbers of vacuoles within individual blastomeres are sufficient to warrant a quality grade 3 score. At least 25% of the embryonic cells should be an intact, viable and cohesive embryonic mass. Multiple extruded cells of widely varying sizes, indicating a prolonged and persistent problem with the cohesiveness of embryonic blastomeres, would cause an embryo to be assigned a quality grade 3 score.

Quality grade code 4: dead or degenerating

Entities that are assigned a quality grade 4 score are nonviable and should not be transferred to recipient females or cryopreserved. Dead/degenerating embryos typically fall in this category because of extremely dark cytoplasm, nonintact cell membranes, and other significant defects. Because embryos typically are recovered on approximately day 7 of the estrous cycle, any embryo that has not advanced to at least the morula stage of embryonic development should be assigned an embryo quality grade 4 score.

Despite the strong correlation that exists between embryo quality grade and post-transfer pregnancy rate, some quality grade 1 embryos fail to produce a pregnancy after transfer whereas some quality grade 3 embryos produce a higher than expected pregnancy rate The IETS Manual20 states:

it should be recognized that visual evaluation of embryos is a subjective valuation of a biological system and is not an exact science. Furthermore, there are other factors such as environmental conditions, recipient quality, and technician capacity that are important in obtaining pregnancies after transfer of embryos.

Embryo evaluation procedure

During the recovery procedure, embryos are typically collected using an embryo filtration device that minimizes the volume of flushing medium through which the technician must search to locate the embryos. Some embryo filtration devices also serve as the embryo search dish, whereas other devices require that embryos be transferred from the filtration device to a Petri dish for searching. After embryos are recovered, they must be located in the recovery medium using a stereomicroscope typically under 10–15× magnification. Embryos are subsequently transferred to a Petri dish containing embryo holding medium where they will be evaluated at a minimum magnification of 50× before being assigned the two-digit IETS code for stage of development and embryo quality.

As illustrated in Figure 79.2, the compact morula, early blastocyst, and blastocyst stages of embryonic development are the ones most commonly recovered on day 7 after estrus. The expanded blastocyst is typically recovered on day 8 but can sometimes be found after a day 7 embryo recovery. The zona pellucida, because of its translucency and ability to refract light, is the landmark structure that helps technicians identify embryos and ova during the search procedure. Once an embryo progresses to the hatched blastocyst stage it may become harder to identify because it may have a collapsed appearance and, without the zona pellucida, it can easily be mistaken for endometrial cells or other cellular debris in the Petri dish.

Although each harvested embryo must be assigned its own two-digit code, there is great value in examining all harvested embryos/ova as a group. This comparative evaluation usually makes it easy for the technician to distinguish UFOs from embryos, as well as to compare embryos that are expanded and have a thinner zona pellucida with embryos that have not yet expanded.

A good predictor of an embryo’s viability is its stage of development relative to what it should be on a given day after estrus. From a superovulated cow, one should expect variety in the stages of embryonic development recovered because there is variation in the time at which ovarian follicles rupture and release their ova. Similarly, one should also expect variation in embryo quality grade. It is not uncommon to obtain from the same donor female excellent-quality embryos in addition to UFOs and degenerate embryos. Based on the American Embryo Transfer Association survey report,21 the average number of viable embryos recovered from dairy donors was 5.7 (representing 56% of the total ova/embryos recovered) and from beef donors was 6.9 (also representing 56% of total ova/embryos recovered). The average number of degenerate embryos and unfertilized ova recovered was 1.6 (14% of total embryos/ova) and 3.1 (29% of total embryos/ova), respectively, from dairy donors and 2.0 (15% of total ova/embryos) and 3.5 (27% of total ova/embryos), respectively, from beef donors.

Practical application of embryo evaluation guidelines

To help technicians learn how to properly evaluate bovine in vivo derived embryos, Figure 79.3 depicts (using photographs of actual bovine ova and embryos) the range in embryonic developmental stage that may be obtained after performing a day 7 embryo recovery. A more developmentally advanced embryo (expanded hatched blastocyst) is shown in Figure 79.3b, although an embryo at this developmental stage is not usually expected on day 7. Many people find the side-by-side photos of actual specimens more helpful for learning embryo grading and classification than illustrations/sketches. In addition to the composite image in Figure 79.3 depicting the range in embryonic developmental stages, the figures in the remainder of this chapter show not only the principles of embryo grading and classification but also some of the common challenges faced by embryo technicians.


Figure 79.3 (a) Comparison of developmental stages of in vivo-derived bovine embryos collected on days 6–8 after estrus. At day 7, a stage code 1 embryo is considered an unfertilized ovum and a stage code 2 embryo is considered dead or degenerate. (b) Expanded hatched blastocyst typically recovered on or after day 8 after estrus; note the empty zona pellucida on the right and the expanded embryo on the left.

(courtesy of Dr Brad Lindsey)

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Aug 24, 2017 | Posted by in GENERAL | Comments Off on Evaluation of In Vivo-Derived Bovine Embryos
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