EMBRYOLOGICAL TERMINOLOGY

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 twocell, four-cell, etc. stages. At the !-cell stage, the embryo becomes a morula "#atin for mulberry$. %hen a cavity "blastocoele$ forms between the cells of the embryo, it is termed a blastocyst. To add further confusion, all of these stages of embryos are fre&uently called eggs or ova. 'mbryos of various stages are illustrated in (igures ) to *+. The first three divisions of the embryo are called cleavage divisions; thus, one-to eightcell embryos are defined as cleavage stages. ,uring this time the embryo actually decreases in weight. -nly at the morula stage does the embryo begin to weigh more than at the one-cell stage.
FIGURE 14 Location of different develop ental !ta"e! of #ovine e #r$o! in reprod%ctive tract &fir!t appeared in 'oard( Dairyman( 1) Marc* 1+,,( p- .4/0

,uring the morula stage, cells of embryos change from spherical to polygonal in shape. This phenomenon is termed compaction. ,uring compaction, specialized .unctions from between cells, so the cells can communicate with each other. (re&uently, compacted morulae are termed tight morulae. /ompacted morulae are smaller than pre-compacted embryos. /ompaction is an e0cellent sign that the embryo is developing normally; lac1 of compaction by si0 days after oestrus in cattle indicates retarded development. As the morula develops into a blastocyst, it forms a cavity, the blastocoele, by e0pending energy to pump fluid between the cells. Thus blastocyst formation also is indicative of continued normal embryonic development. /onversely, lac1 of blastocoele formation by seven to eight days after oestrus in cattle signifies retarded development.
FIGURE 11 2ia"ra of nor al #ovine e #r$o!

The zona pellucida is a gelatin-li1e capsule that surrounds the oocyte and early embryo. It has receptors for sperm that are inactivated after fertilization, it 1eeps 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. %hen the blastocoele becomes very large, the embryo e0pands "normally eight to nine days after oestrus$, which thins the zona pellucida. This is the e0panded blastocyst stage. After one to one days more, the e0pansion is so great that the embryo hatches out of the zona

pellucida, perhaps aided by enzymes. 2atched blastocysts become elipsoid in shape 3 4 days after oestrus, and then elongate mar1edly by )3 ! days post-oestrus. 5y day 63 7 the embryo elongates sufficiently to reach the tip of both uterine horns.

E3ALUATION
(or many beginners, the most intimidating aspect of the embryo transfer process is morphological evaluation of embryos. -bviously, there is no profit in transferring unfertilized ova or degenerate embryos, nor in discarding perfectly normal ones. 5oth errors are common when people are first gaining e0perience, and not infre&uent when more seasoned personnel ma1e hasty decisions. There are three elements to successful evaluation of embryos8 training, e0perience and proper e&uipment. Training includes learning the correct morphology of embryos at different times postoestrus and the meaning of deviations from normal morphology. -ne must also learn how to manipulate and e0amine embryos. '0perience is gained by e0amining many embryos at different stages of development. Ideally hundreds of embryos should be studied under the guidance of someone e0perienced in this area. 9hotographs, drawings or slides of various 1inds of embryos are very useful. 2owever, they can only substitute partially for real embryos. '0perienced personnel can evaluate more than 7+ percent of ova accurately with a good stereomicroscope at 4:; to ):; magnification or less. 2owever, a small percentage of embryos re&uire a compound microscope "at least a :; ob.ective with <; to *:; eyepieces$ for accurate evaluation. (or learning purposes, a compound microscope is especially useful. =ost compound microscopes are poorly designed to e0amine embryos, and wor1ing distance "distance from the embryo to the ob.ective lens$ is fre&uently short. These limitations ma1e it easy to spill the dish containing embryos and to contaminate the fluid containing the embryos with the ob.ective lens. FIGURE 1/ &A0 Follic%lar ooc$te 4it* ad*erent follicle cell!- No ar!5i optic!- &B0 Follic%lar ooc$te after re ovin" follicle cell!- No ar!5i optic!- &C0 Nor al appearin" 16cell ov% recovered five da$! after oe!tr%!- Note !per ato7oa in t*e 7ona pell%cidaBri"*t6field optic!- &20 Nor al( %nfertili7ed( ov%lated ooc$te recovered t*ree da$! after oe!tr%!- Bri"*t6field optic!- &Fi"%re! 1/B( 1+C( .)B( .)C and .8B fir!t appeared in Science( .119 811:81,( cop$ri"*t( AAA;( 1+,10

'mbryos collected si0 days post-oestrus should be post-compaction or so-called tight morulae. They should have +:3<: cells. Although it is impossible to count cells accurately in post-compaction embryos without resorting to procedures that damage embryos, it is useful to ma1e estimates of cell numbers. 'mbryos should be generally spherical or ovoid, not too light nor too dar1 in colour "(igure * 5 and / illustrates unacceptable e0tremes$, and have uniform cell size. ,eviations from normal include irregular cell sizes, large vacuoles in cells, areas of degeneration in the embryos, some cells not compacted with the main cell mass "termed e0truded or e0cluded blastomeres$, and a damaged zona pellucida. >early *:34: percent of good embryos have some detectable morphological abnormality such as a few e0cluded blastomeres. =ost of these abnormalities are a matter of degree. If part of the embryo appears degenerate, but the bul1 of the embryo appears normal, it has an e0cellent chance of developing into a normal calf "e.g. (igure **5$; morphologically abnormal embryos do not result in abnormal calves. >ote that pregnancy rates with bisected embryos "see /hapter :$ are really &uite good, which means that half of the cells can be degenerate without mar1edly lowering pregnancy rates. FIGURE 1< &A0 Unfertili7ed ooc$te recovered five da$! after oe!tr%!- No ar!5i optic!- &B0

;a e ov% a! in &A0 4it* #ri"*t6field optic!- &C0 Crac5ed( e pt$ 7ona pell%cida recovered five da$! after oe!tr%!- No ar!5i optic!- &20 Unfertili7ed ooc$te recovered !i= da$! after oe!tr%!- Note #li!ter! of clear c$topla! - No ar!5i optic!

FIGURE 1, &A0 2e"enerate( %nfertili7ed ov% recovered five da$! after oe!tr%!- No ar!5i optic!- &B0 Unfertili7ed ov% 4it* t4o fra" ent! of c$topla! - Note lar"e ve!icle! 4it*in c$topla! - Bri"*t6field optic!- &C0 Fra" ented ov% ( li5el$ %nfertili7ed recovered five da$! after oe!tr%!- Bri"*t6field optic!- &20 2i!inte"rated ov% ( pro#a#l$ %nfertili7ed- Bri"*t6field optic!

FIGURE 1+ &A0 Nor al appearin" .6cell e #r$o recovered fo%r and a *alf da$! after oe!tr%!( #ri"*t6field optic!- &B0 2e"eneratin" .6cell e #r$o recovered five da$! after oe!tr%!- Note clear c$topla! in one #la!to ere- &C0 Nor al 46cell e #r$o recovered t4o and a *alf da$! after oe!tr%!- No ar!5i optic!- &20 A .6cell e #r$o recovered five da$! after oe!tr%!- Note clear c$topla! - No ar!5i optic!

,ay-6 embryos should be early blastocysts. As mentioned earlier, presence of a blastocoelic cavity is a good sign. ,ay-< embryos should have a large blastocoele and some should be e0panding, i.e. the diameter should be increasing so that the zona pellucida is thinned. A distinct, inner cell mass should be present. -ther aspects of morphology should be as described earlier in this section. As with day-! embryos, various imperfections are not uncommon in perfectly acceptable embryos. FIGURE .) &A0 Nor al ,6cell e #r$o recovered t*ree da$! after oe!tr%!- Bri"*t6field optic!&B0 ;a e e #r$o a! in &A0 #%t No ar!5i optic!- &C0 Nor al 1.6 to 146cell e #r$o recovered fo%r da$! after oe!tr%!- No ar!5i optic!- &20 ;everel$ retarded 1.6 to 146cell e #r$o recovered !i= da$! after oe!tr%!- Bri"*t6field optic!-

FIGURE .1 &A0 Unco pacted or%la recovered t*ree da$! after oe!tr%!( pro#a#l$ de"eneratin"- &B0 Unco pacted or%la recovered t*ree da$! after oe!tr%!> dar5 c$topla! - &C0 ;everel$ retarded and de"eneratin" e #r$o recovered !i= da$! after oe!tr%!- &20 ;everel$ de"enerate e #r$o recovered !even da$! after oe!tr%!All are #ri"*t6field optic!-

In our laboratory at /olorado ?tate University, we have evaluated nearly + ::: bovine ova over the years. About one-third of these have been unfertilized or severely degenerate; perhaps the most important tas1 in evaluating ova is to identify these and fail to transfer them so that pregnancy rates are not lowered. The single most difficult tas1 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$, which can loo1 very similar in size and te0ture. The unfertilized ovum has a perfectly smooth cell membrane, at least over a part of the cell, while the tight morula will have a slightly scalloped appearance. FIGURE .. &A0 Ne4l$ co pacted or%la recovered !even da$! after oe!tr%!- Bri"*t6field optic!- &B0 Co pacted or%la recovered !even da$! after oe!tr%! 4it* !everal e=cl%ded cell!> "ood orp*olo"ical ?%alit$- No ar!5i optic!- &C0 Co pacted or%la recovered !even and a *alf da$! after oe!tr%! 4it* an$ lar"e( e=cl%ded cell!( fair orp*olo"ical ?%alit$- Bri"*t6field optic!- &20 @oor ?%alit$ or%la 4it* an$ de"enerate cell!- 'o4ever( t*e ! all( co pacted a!! to t*e lo4er left *a! a ! all c*ance of developin" into a calf- Bri"*t6field optic!

FIGURE .8 &A0 Nor al( earl$( e=panded #la!toc$!t recovered !even da$! after oe!tr%!Bri"*t6field optic!- &B0 ;a e e #r$o a! in &A0 #%t No ar!5i optic!- &C0 Nor al( e=panded #la!toc$!t recovered !even and a *alf da$! after oe!tr%!- Note t*e t*inned 7ona pell%cida- Bri"*t6field optic!- &20 'atc*in" #la!toc$!t t$picall$ fo%nd nine da$! after oe!tr%!- Bri"*t6field optic!

%ith e0perience, these two types of ova can be distinguished easily, especially with a compound microscope "(igures *) and *+$. -ccasionally they are classified incorrectly, even by e0perts who do not ta1e sufficient time "really only +3 : seconds$ to evaluate the embryos correctly. A second, much more rare misclassification occurs when unfertilized ova degenerate in the centre and become &uite clear, resembling a blastocyst at first glance "(igure *+,$. =ost other misclassifications are a matter of

degree in distinguishing among good, fair and poor embryos "see below$. An e0cellent treatise on ovine embryo morphology is authored by %intenberger Torres and ?evellec, 7<6. 5ovine and ovine embryos are nearly identical morphologically. FIGURE .4 &A0 Good ?%alit$ co pacted or%la 4it* a fe4 de"enerate cell! recovered !i= and a *alf da$! after oe!tr%!- Bri"*t6field optic!- &B0 Unfertili7ed ov% recovered !even da$! after oe!tr%!( ea!il$ i!ta5en for a or%la 4it* a di!!ectin" icro!cope- Bri"*t6field optic!- &C0 2e"enerate( pro#a#l$ %nfertili7ed ov% ( can #e i!ta5en for or%la at lo4er a"nification- &20 2e"enerate( %nfertili7ed ov% ( ea!il$ i!ta5en for or%la at lo4er a"nification

FIGURE .1 &A0 Ne4l$ co pacted or%la recovered !i= da$! after oe!tr%! &"ood ?%alit$0 #%t 4it* one lar"e and pro#a#l$ a#nor al cell to t*e %pper ri"*t- &B0 Unfertili7ed ov% ea!il$ i!ta5en for a or%la- &C0 Nor al #la!toc$!t recovered !even and a *alf da$! after oe!tr%!- &20 Unfertili7ed ov% 4it* lar"e ve!icle recovered five da$! after oe!tr%!( ea!il$ i!ta5en for a #la!toc$!t at lo4er a"nificationAll are #ri"*t6field optic!

TA5#' + ;ta"e of nor al e #r$onic develop ent a! a f%nction of da$! after donorA! oe!tr%!
?tage of ,ays after onset of oestrus development -cell *-cell )-cell <-cell !-cell Tight morula 'arly blastocyst 5lastocyst :3* 34 *34 43+* )3+* +36 63< 637

'arly morula +3!

'0panded blastocyst 2atching blastocyst

<3 : 73

@ 'mbryos usually move from the oviduct to the uterus at the <- to !-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. 'ach ovum should then be carefully e0amined individually by focusing up and down and in certain cases rolling the embryo with a pipette or by sha1ing 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 e0amined with a compound microscope. In our laboratory, we classify all ova into si0 categories. (or those frozen or transferred, the final classification is generally made .ust before freezing or transfer. %e fill out a form before ma1ing the final classification, which forces the evaluator to record "and thus ta1e into account$ the following criteria8 age "days post-oestrus$, cell number, compaction status, variability in cell size, colour of cytoplasm, areas of degeneration, numbers of e0cluded blastomeres, size of peri-vitelline space, stage of embryonic development, and number of days the embryo is retarded from normal "e.g. a four-cell embryo recovered five days after oestrus would be two days retarded$. After these are recorded, ova are placed into one of the following si0 categories8 TA5#' ! @re"nanc$ rate! of e #r$o! cla!!ified into ?%alit$ "ro%p! #a!ed on "ro!! orp*olo"$
/lassification >o. '0cellent Aood (air 9oor
a, b, c

9ercentage of embryos

9regnancy rate !4a +<a 4

b

*6+ +) +* 4: )* )* < <

*c

9regnancy rates with different superscripts differ, 9B:+.

'0cellent8 perfect embryo for its age Aood8 trivial imperfections such as oval zona pellucida, a few, small e0cluded cells, or slightly asymmetrical shape (air8 definite but not severe abnormalities such as moderate numbers of e0cluded cells, small size, small amounts of degeneration, or retarded in development by up to one day 9oor8 considerable degeneration, vesiculated cells, greatly varying cell size, failure of compaction, very small andCor retarded by two days in development ,egenerate8 severely degenerate and not worth transferring

Unfertilized or two- or three-cell.

It is often impossible to determine if an ovum is a severely degenerate embryo or is unfertilized. 'ven two-or three-cell embryos may in fact be fragmented, unfertilized ova. Table ! "from 'lsden et al., 76<$ provides a distribution of embryos into the four categories considered transferable as well as pregnancy rates for each group. /learly, this classification system ran1s embryos reasonably well on a statistical basis. -f 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 e0cellent embryos are suitable for splitting, and only fair, good, and e0cellent ones are suitable for freezing. Desults of freezing fair &uality embryos are marginal.