In mammals, the female gamete is called an egg or ovum; the correct technical term for the newly ovulated female gamete is an oocyte. Upon fertilization, the oocyte becomes a one-cell embryo, sometimes referred to as a zygote. The embryo then divides into two-cell, four-cell, etc. stages. At the 16-cell stage, the embryo becomes a morula (Latin for mulberry). When a cavity (blastocoele) forms between the cells of the embryo, it is termed a blastocyst. To add further confusion, all of these stages of embryos are frequently called eggs or ova. Embryos of various stages are illustrated in Figures 14 to 25.
The first three divisions of the embryo are called cleavage divisions; thus, one-to eight-cell embryos are defined as cleavage stages. During this time the embryo actually decreases in weight. Only at the morula stage does the embryo begin to weigh more than at the one-cell stage.
Location of different developmental stages of bovine embryos in reproductive tract (first appeared in Hoard, Dairyman, 10 March 1988, p. 246)
During the morula stage, cells of embryos change from spherical to polygonal in shape. This phenomenon is termed compaction. During compaction, specialized junctions from between cells, so the cells can communicate with each other. Frequently, compacted morulae are termed tight morulae. Compacted morulae are smaller than pre-compacted embryos. Compaction is an excellent sign that the embryo is developing normally; lack of compaction by six days after oestrus in cattle indicates retarded development.
As the morula develops into a blastocyst, it forms a cavity, the blastocoele, by expending energy to pump fluid between the cells. Thus blastocyst formation also is indicative of continued normal embryonic development. Conversely, lack of blastocoele formation by seven to eight days after oestrus in cattle signifies retarded development.
Diagram of normal bovine embryos
The zona pellucida is a gelatin-like capsule that surrounds the oocyte and early embryo. It has receptors for sperm that are inactivated after fertilization, it keeps the cells of the pre-compaction embryo together, and protects these young cells from the immune system and from pathogens. If the zona pellucida is removed from pre-compaction embryos, the cells come apart upon embryo transfer and then degenerate. When the blastocoele becomes very large, the embryo expands (normally eight to nine days after oestrus), which thins the zona pellucida. This is the expanded blastocyst stage. After one to one days more, the expansion is so great that the embryo hatches out of the zona pellucida, perhaps aided by enzymes. Hatched blastocysts become elipsoid in shape 11–13 days after oestrus, and then elongate markedly by 14–16 days post-oestrus. By day 17–19 the embryo elongates sufficiently to reach the tip of both uterine horns.
For many beginners, the most intimidating aspect of the embryo transfer process is morphological evaluation of embryos. Obviously, there is no profit in transferring unfertilized ova or degenerate embryos, nor in discarding perfectly normal ones. Both errors are common when people are first gaining experience, and not infrequent when more seasoned personnel make hasty decisions. There are three elements to successful evaluation of embryos: training, experience and proper equipment.
Training includes learning the correct morphology of embryos at different times post-oestrus and the meaning of deviations from normal morphology. One must also learn how to manipulate and examine embryos. Experience is gained by examining many embryos at different stages of development. Ideally hundreds of embryos should be studied under the guidance of someone experienced in this area. Photographs, drawings or slides of various kinds of embryos are very useful. However, they can only substitute partially for real embryos. Experienced personnel can evaluate more than 95 percent of ova accurately with a good stereomicroscope at 30X to 40X magnification or less. However, a small percentage of embryos require a compound microscope (at least a 10X objective with 8X to 20X eyepieces) for accurate evaluation. For learning purposes, a compound microscope is especially useful. Most compound microscopes are poorly designed to examine embryos, and working distance (distance from the embryo to the objective lens) is frequently short. These limitations make it easy to spill the dish containing embryos and to contaminate the fluid containing the embryos with the objective lens.
(A) Follicular oocyte with adherent follicle cells. Nomarski optics. (B) Follicular oocyte after removing follicle cells. Nomarski optics. (C) Normal appearing 1-cell ovum recovered five days after oestrus. Note spermatozoa in the zona pellucida. Bright-field optics. (D) Normal, unfertilized, ovulated oocyte recovered three days after oestrus. Bright-field optics. (Figures 16B, 19C, 20B, 20C and 23B first appeared in Science, 211: 351–358, copyright, AAAS, 1981)
Embryos collected six days post-oestrus should be post-compaction or so-called tight morulae. They should have 50–80 cells. Although it is impossible to count cells accurately in post-compaction embryos without resorting to procedures that damage embryos, it is useful to make estimates of cell numbers. Embryos should be generally spherical or ovoid, not too light nor too dark in colour (Figure 21B and C illustrates unacceptable extremes), and have uniform cell size. Deviations from normal include irregular cell sizes, large vacuoles in cells, areas of degeneration in the embryos, some cells not compacted with the main cell mass (termed extruded or excluded blastomeres), and a damaged zona pellucida. Nearly 20–30 percent of good embryos have some detectable morphological abnormality such as a few excluded blastomeres. Most of these abnormalities are a matter of degree. If part of the embryo appears degenerate, but the bulk of the embryo appears normal, it has an excellent chance of developing into a normal calf (e.g. Figure 22B); morphologically abnormal embryos do not result in abnormal calves. Note that pregnancy rates with bisected embryos (see Chapter 10) are really quite good, which means that half of the cells can be degenerate without markedly lowering pregnancy rates.
(A) Unfertilized oocyte recovered five days after oestrus. Nomarski optics. (B) Same ovum as in (A) with bright-field optics. (C) Cracked, empty zona pellucida recovered five days after oestrus. Nomarski optics. (D) Unfertilized oocyte recovered six days after oestrus. Note blisters of clear cytoplasm. Nomarski optics
(A) Degenerate, unfertilized ovum recovered five days after oestrus. Nomarski optics. (B) Unfertilized ovum with two fragments of cytoplasm. Note large vesicles within cytoplasm. Bright-field optics. (C) Fragmented ovum, likely unfertilized recovered five days after oestrus. Bright-field optics. (D) Disintegrated ovum, probably unfertilized. Bright-field optics
(A) Normal appearing 2-cell embryo recovered four and a half days after oestrus, bright-field optics. (B) Degenerating 2-cell embryo recovered five days after oestrus. Note clear cytoplasm in one blastomere. (C) Normal 4-cell embryo recovered two and a half days after oestrus. Nomarski optics. (D) A 2-cell embryo recovered five days after oestrus. Note clear cytoplasm. Nomarski optics
Day-7 embryos should be early blastocysts. As mentioned earlier, presence of a blastocoelic cavity is a good sign. Day-8 embryos should have a large blastocoele and some should be expanding, i.e. the diameter should be increasing so that the zona pellucida is thinned. A distinct, inner cell mass should be present. Other aspects of morphology should be as described earlier in this section. As with day-6 embryos, various imperfections are not uncommon in perfectly acceptable embryos.
(A) Normal 8-cell embryo recovered three days after oestrus. Bright-field optics. (B) Same embryo as in (A) but Nomarski optics. (C) Normal 12- to 14-cell embryo recovered four days after oestrus. Nomarski optics. (D) Severely retarded 12- to 14-cell embryo recovered six days after oestrus. Bright-field optics.
(A) Uncompacted morula recovered three days after oestrus, probably degenerating. (B) Uncompacted morula recovered three days after oestrus; dark cytoplasm. (C) Severely retarded and degenerating embryo recovered six days after oestrus. (D) Severely degenerate embryo recovered seven days after oestrus.
All are bright-field optics.
In our laboratory at Colorado State University, we have evaluated nearly 15 000 bovine ova over the years. About one-third of these have been unfertilized or severely degenerate; perhaps the most important task in evaluating ova is to identify these and fail to transfer them so that pregnancy rates are not lowered. The single most difficult task for people learning to classify embryos is to distinguish between tight morulae and unfertilized oocytes (note that unfertilized embryo is improper terminology and internally contradictory), which can look very similar in size and texture. The unfertilized ovum has a perfectly smooth cell membrane, at least over a part of the cell, while the tight morula will have a slightly scalloped appearance.
(A) Newly compacted morula recovered seven days after oestrus. Bright-field optics. (B) Compacted morula recovered seven days after oestrus with several excluded cells; good morphological quality. Nomarski optics. (C) Compacted morula recovered seven and a half days after oestrus with many large, excluded cells, fair morphological quality. Bright-field optics. (D) Poor quality morula with many degenerate cells. However, the small, compacted mass to the lower left has a small chance of developing into a calf. Bright-field optics
(A) Normal, early, expanded blastocyst recovered seven days after oestrus. Bright-field optics. (B) Same embryo as in (A) but Nomarski optics. (C) Normal, expanded blastocyst recovered seven and a half days after oestrus. Note the thinned zona pellucida. Bright-field optics. (D) Hatching blastocyst typically found nine days after oestrus. Bright-field optics
With experience, these two types of ova can be distinguished easily, especially with a compound microscope (Figures 24 and 25). Occasionally they are classified incorrectly, even by experts who do not take sufficient time (really only 5–10 seconds) to evaluate the embryos correctly. A second, much more rare misclassification occurs when unfertilized ova degenerate in the centre and become quite clear, resembling a blastocyst at first glance (Figure 25D). Most other misclassifications are a matter of degree in distinguishing among good, fair and poor embryos (see below). An excellent treatise on ovine embryo morphology is authored by Wintenberger Torres and Sevellec, 1987. Bovine and ovine embryos are nearly identical morphologically.
(A) Good quality compacted morula with a few degenerate cells recovered six and a half days after oestrus. Bright-field optics. (B) Unfertilized ovum recovered seven days after oestrus, easily mistaken for a morula with a dissecting microscope. Bright-field optics. (C) Degenerate, probably unfertilized ovum, can be mistaken for morula at lower magnification. (D) Degenerate, unfertilized ovum, easily mistaken for morula at lower magnification
(A) Newly compacted morula recovered six days after oestrus (good quality) but with one large and probably abnormal cell to the upper right. (B) Unfertilized ovum easily mistaken for a morula. (C) Normal blastocyst recovered seven and a half days after oestrus. (D) Unfertilized ovum with large vesicle recovered five days after oestrus, easily mistaken for a blastocyst at lower magnification.
All are bright-field optics
Stage of normal embryonic development as a function of days after donor's oestrus
|Stage of development||Days after onset of oestrus|
* Embryos usually move from the oviduct to the uterus at the 8- to 16-cell stage.
The proper procedure for classifying embryos is to isolate them, remove debris (which occurs automatically in the process of washing them three times), and then separate them into groups of transferable (or freezable or splittable) and non-transferable (unfertilized or severely degenerate) groups. Each ovum should then be carefully examined individually by focusing up and down and in certain cases rolling the embryo with a pipette or by shaking the dish. Those classified incorrectly should be placed in the proper groups and the non-transferable ova set aside. In cases in which classification is uncertain, ova should be examined with a compound microscope.
In our laboratory, we classify all ova into six categories. For those frozen or transferred, the final classification is generally made just before freezing or transfer. We fill out a form before making the final classification, which forces the evaluator to record (and thus take into account) the following criteria: age (days post-oestrus), cell number, compaction status, variability in cell size, colour of cytoplasm, areas of degeneration, numbers of excluded blastomeres, size of peri-vitelline space, stage of embryonic development, and number of days the embryo is retarded from normal (e.g. a four-cell embryo recovered five days after oestrus would be two days retarded). After these are recorded, ova are placed into one of the following six categories:
Pregnancy rates of embryos classified into quality groups based on gross morphology
|Classification||No.||Percentage of embryos||Pregnancy rate|
a, b, c Pregnancy rates with different superscripts differ, P<05.
It is often impossible to determine if an ovum is a severely degenerate embryo or is unfertilized. Even two-or three-cell embryos may in fact be fragmented, unfertilized ova. Table 6 (from Elsden et al., 1978) provides a distribution of embryos into the four categories considered transferable as well as pregnancy rates for each group. Clearly, this classification system ranks embryos reasonably well on a statistical basis. Of course, it is far from ideal from the standpoint of sorting embryos into the group that will result in calves and the group that will not. As a rule of thumb, only good and excellent embryos are suitable for splitting, and only fair, good, and excellent ones are suitable for freezing. Results of freezing fair quality embryos are marginal.