Photographic Study of Chick Embryo Development In vitro1

B. E. DUNN and M. A. BOONE

Department of Poultry Science, Clemson University, Clemson, South Carolina 29631

(Received for publication August 5, 1977)

ABSTRACT A photographic study of development of chick embryos cultured in vitro was conducted in order to demonstrate the relationship between embryos and extraembryonic membranes during culture. Survival of embryos in this system has been extended to 21 days of total incubation (3 days in shell plus 18 days in vitro) and development to Hamburger-Hamilton stage 45 (partial or complete yolk sac retraction). A few mature cultured embryos have "pipped" through the chorioallantoic membrane and have emitted audible peeping.

Three-day embryos and egg contents were cultured in commercial plastic wrap suspended in plastic tripods. The yolk sac expanded and flattened to cover the bottom of the culture chamber by five days of total incubation. Downgrowth of the yolk sac vascular system along the plastic wrap walls of the chamber began by six days, while downgrowth of the chorioallantoic membrane (CAM) began by eight days. The CAM often completely enclosed cultured egg contents by 17 days, an event which occurs in ovo by 11 to 12 days. Lack of formation of the albumen sac resulted in inhibition of albumen uptake during the latter portion of incubation.

A small number of embryos were on their right side rather than on the normal left side during the first two days of culture. Many of the right side cultured embryos experienced "compensatory torsion" onto their left side by three days of culture (six days of total incubation).

INTRODUCTION

Recent success in long-term culture of the chick embryo in vitro (Dunn, 1974; Dunn and Boone, 1976) has extended the survival of cultured embryos to 21 days of total incuba­tion (3 days in shell plus 18 days in culture) and the max imum developmental stage to Hamburger-Hamilton Stage 45 , at which time partial or complete yolk sac retract ion occurs (Hamilton, 1952) .

In previous studies, growth of cultured embryos has been assessed on the basis of developmental stage at t ime of death (Dunn and Boone, 1976) and on the basis of incuba­tion age of living embryos (Dunn and Boone, 1971) . Growth of embryos in this system is fairly normal through 12 days of incubat ion. Beyond 12 days, however, cultured embryos exhibit significantly depressed weight, toe length, and mineral con ten t (Dunn and Boone, 1977) .

Shell-less culture techniques have recently been applied in chorioallantoic grafting of central nervous tissue (Corner and Richter, 1973) and in studies of host-mediated tumor

1 Published with the approval of the Director of the South Carolina Agricultural Experiment Station as technical contribution No. 1460.

vacular response (Auerbach et al., 1975; Klags-brun et al, 1976) . A brief photographic record of development of embryos cultured in petri dishes has appeared (Auerbach et al., 1974) .

The technique used in this s tudy allows approximate ly 90% of embryos cultured to be maintained for up to 14 days of total incuba­tion (Dunn and Boone, 1976) , about twice the survival rate in petri dishes. The technique facilitates rapid expansion and more normal downgrowth of ext raembryonic membranes around the egg contents than observed in petri dishes. The purpose of this paper, then, is to provide a photographic account of the growth and relationship of the embryo and extraem­bryonic membranes in a new culture system.

MATERIALS AND METHODS

All embryos to be cultured were incubated for three days within shells under standard condit ions of tempera ture , humidi ty , and turn­ing prior to transfer to culture chambers. Culture me thods used were those described previously (Dunn and Boone, 1976). Briefly, egg contents were suspended in commercial sandwich-type plastic wrap in specially con­structed plastic t r ipods. Tripods were covered with the top half of a 100 X 15 mm sterile disposable petri dish raised slightly to allow gas exchange. Embryos were maintained at satura-

1978 Poultry Sci 57:370-3 77 3 70

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CULTURED CHICK EMBRYOS 371

tion humidi ty with 1 — 2% C 0 2 in air in a Forma C 0 2 incubator . Photographs were taken using Kodak Panatomic X film in a Honeywell Pentax Spotmat ic F camera mounted on a portable light stand. Culture chambers with developing embryos were removed from incuba­tors for photography. The petri dish covers were removed from the chambers to eliminate reflection, then were replaced prior to returning chambers to the incubators.

For comparat ive purposes, control egg con­tents were transferred from shells after four to eight days of incubation to the type of chamber used to culture embryos . Approximate ly 15 to 20 ml of warm Lock's balanced saline solution was introduced into the plastic wrap prior to cracking the shells. Addit ion of the saline solution was necessary to float the yolks and to prevent damage to them during manipulat ion. No saline solution was added to cultured egg contents .

RESULTS AND DISCUSSION

Comparison of Development In ovo and In vitro. Day 3 (Fig. 1). After three days of incubat ion in the shell the embryo is easily discernible upon the yolk surface. By this t ime

FIG. 1. Egg contents after three days of incubation within the shell. The embryo (e) is located centrally. The area vasculosa (v) of the yolk sac is bounded by the sinus terminalis (large solid arrows). Albumen (al) surrounds the yolk. The inner light ring (t) is the tripod which suspends plastic wrap held in place by the outer dark split ring (s). Magnification of Fig. 1—11 and 15 can be determined based on inner diameter of tripod = 7.8 cm. (thin arrows).

torsion of the embryonic body has advanced such that the cephalic port ion lies on its left side upon the yolk, but the caudal port ion posterior to the heart is not completely turned on its side (Pat ten, 1971). The lateral vessels and sinus terminalis of the yolk sac vascular system are well developed. Albumen surrounds the yolk in the culture chamber . Not all three-day yolks retain a spherical shape upon transfer to cul ture chambers. Often they are elliptical or irregular in shape, probably due to weakening of the vitelline membrane during the first several days of incubation (Rol 'nik, 1968). However, embryonic survival is generally not affected by irregularities in yolk conformat ion unless rupture of the yolk sac occurs.

Day 4 (Fig. 2, 3). The yolk flattens out and covers most of the chamber surface after one day in culture (Fig. 2). Flattening and expan­sion of the yolk are a t t r ibuted to the reduced strength of the vitelline membrane and to rapid influx of water from the surrounding a lbumen. Irregular indentat ions in the margin of the yolk may occur in the first 24 hr of culture even in originally spherical yolk (Fig. 2). Such indenta­t ions are probably caused by rup ture of the vitelline membrane , which ruptures in ovo and slips down toward the distal port ion of the yolk sac by three to four days (Romanoff, 1960) . Similar irregularities have been produced exper-

FIG. 2. Cultured egg contents after four days of total incubation. Embryo (e) with small allantois (clear arrow) is at center surrounded by area vasculosa (v) and sinus terminalis (large solid arrows). Small outer solid arrows indicate irregularities in yolk margin. Albumen (al) surrounds the yolk.

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3 72 B. E. DUNN AND M. A. BOONE

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FIG. 3. Control egg contents after four days of incubation in ovo. Abbreviations and arrows as in Fig. 2. Note regular outline of yolk and somewhat larger area vasculosa than in Fig. 2.

imentally in yolks after three to four days of incubation within the shell by pricking the vitelline membrane with a fine needle (Vogelaar and van dcr Boogert, 1925) . No such irregulari­ties are present in the four-day control yolk (Fig. 3). The yolk sac vascular system of four-day cultured embryos is commonly some­what smaller, but more densely vascularized than in control embryos of the same age. By four days, torsion of the embryo in ovo is complete and the embryo lies with its left side entirely on the yolk (Patten, 1971) . Most cultured embryos have undergone torsion and lie completely on their left side by four days, al though except ions do occur (cf. Fig. 16, 18).

Day 5 (Fig. 4, 5). By five days of total incubation the yolk expands nearly to the border of the support ing plastic wrap (Fig. 4) . In contrast , the shape of the five-day control yolk remains fairly spherical (Fig. 5). The area vasculosa in bo th cultured and control embryos covers a lmost the entire visible por t ion of the yolk. The allantois begins to fuse with the overlying t ransparent chorion to form the chorioallantoic membrane (CAM) at this t ime.

Day 6 (Fig. 6, 7) . By six days of total incubat ion (Fig. 6) the yolk has expanded to contac t the support ing plastic wrap completely along its periphery and conforms to the many folds and indenta t ions in the plastic wrap. As a result, the underlying a lbumen is no longer visible. The highly branched area vasculosa

FIG. 4. Cultured egg contents after five days of total incubation. Midbrain (m) of embryo (e) is located toward top of photo. The allantois (clear arrows) is readily visible. The outer margin of the area vasculosa (dark arrows) extends nearly to the support­ing plastic wrap.

covers the entire yolk surface and some blood vessels begin to grow down the sides of the yolk bounded by the plastic wrap. The yolk of a six-day control embryo is still fairly spherical (Fig. 7). The vascular por t ion of the control yolk sac ex tends approximate ly to the equator of the yolk (cf. Romanoff, 1960) . The CAM in

FIG. 5. Control egg contents after five days of incubation in ovo. Abbreviations and arrows as in Fig. 4. Yolk outline is almost circular. Albumen and saline solution surround the yolk.

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CULTURED CHICK EMBRYOS 3 73

FIG. 6. Cultured egg contents after six days of total incubation. Arrows indicate margins of CAM. The yolk sac vascular system extends completely across the chamber surface and has begun to grow down the plasric wrap.

bo th types of embryo is several times the size of the embryo , which is normal at this age in ovo (Romanoff, 1960) .

Day 8 (Fig. 8, 9). During the subsequent two days of incubation the CAM undergoes rapid expansion such that it nearly covers the entire chamber surface area in vitro (Fig. 8) and encloses the upper most half of the yolk in ovo

FIG. 7. Control egg contents after 6 days of incubation in ovo. Arrows indicate limits of CAM. The yolk is circular in outline with less visible surface area than in the cultured yolk in Fig. 6.

FIG. 8. Cultured egg contents after eight days of total incubation. The CAM (arrows) almost complete­ly covers the underlying yolk. Characteristic embry­onic movements are readily observed at this time. Fig. 1, 2, 4, 6, and 8 are photographs of the same cultured embryo at different ages.

(Fig. 9). Downgrowth of the CAM along the plastic wrap generally begins at this t ime.

Day 13 (Fig. 10). By 13 days of total incubation the major blood vessels of the CAM are prominent . They undula te not iceably with the flow of blood. Down has formed on the embryo . Vigorous movements of the head and legs are common . The underlying yolk no

FIG. 9. Control egg contents after eight days of incubation in ovo. Arrows indicate extent of the CAM.

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3 74 B. E. DUNN AND M. A. BOONE

FIG. 10. Cultured egg contents after 13 days of total incubation. Prominent major vessels (a) of the CAM overlie the embryo (e) and egg contents. Size of the yolk is much reduced from its size at eight days of total incubation (Fig. 8).

FIG. 11. Cultured egg contents after 20 days of total incubation. The embryo had just died. Regression of the CAM is apparent. The yolk sac (y) is visible. The upper arrow indicates right eye of the embryo. The beak had punctured the overlying CAM (lower arrow).

longer extends to the confines of the plastic wrap but is much reduced in size. Cultured embryos remain on the surface of the yolk rather than becoming enveloped by it as in ovo.

Day 15—16. The allantoic fluid frequently becomes cloudy after 15 to 16 days of total incubation due t o the presence of insoluble metabolic waste products . Visibility of the embryo is concomitant ly reduced.

Day 20 (Fig. 11). Figure 11 shows a 20-day embryo which died immediately prior to this observation. The CAM circulatory system has undergone marked regression and hemorrhag­ing had occurred. The embryo punctured ( "p ipped") a hole through the overlying CAM with its beak. Other cul tured embryos have been observed which " p i p p e d " through the CAM at 19.5 to 20.5 days of total incubation and emit ted clearly audible peeping sounds prior to their death within culture chambers. Complete retract ion of the yolk sac by cultured embryos has also been observed (Dunn and Boone, 1976) .

Fig. 12 is a comparison of control and cultured embryos after 18 days of total incuba­t ion. Both embryos were alive prior to the t ime at which the p h o t o was taken. While the general body conformity and right third toe length (19.5 m m ) of the cultured embryo compared favorably with the control (20 m m ) , wet weight

of the former was abou t 60% tha t of the control , similar to results noted previously (Dunn and Boone, 1977) .

Membrane Downgrowth. As noted above, downgrowth of the area vasculosa of the yolk sac along the plastic wrap begins by six days, while downgrowth of the CAM begins by eight to nine days. By 10 days both membranes have grown down approximate ly 1 to 2 cm along the

FIG. 12. Comparison of control (a) and cultured (b) embryos after 18 days of total incubation (at which time both embryos were alive). Note similar body conformation, down development and right third toe length. Bar equals 1 cm.

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CULTURED CHICK EMBRYOS 375

plastic wrap. By 14 days (Fig. 13), most of the yolk is surrounded by the ex t raembryonic membranes , leaving only a small por t ion of the yolk uncovered. This unenclosed area of the yolk apparent ly corresponds to the yolk sac umbilicus in ovo (cf. Romanoff, 1960) . Com­plete enclosure of the yolk sac and CAM around the yolk and embryo in culture general­ly occurs by 17 days of total incubation (Fig. 14). In ovo the CAM completely surrounds the egg contents by 11 to 12 days concomi tan t with formation of the albumen sac, a s t ructure associated with transfer of a lbumen into the amniot ic cavity (Romanoff, 1960) . No a lbumen sac forms in vitro, however. The CAM tends to grow over the top of the albumen in culture, rather than enveloping it as in ovo.

Yolk Sac Rupture. It has previously been observed (Dunn and Boone, 1976) tha t em­bryonic morta l i ty is slight up to day 14, bu t increases thereafter. One event associated with increased mortal i ty is rupture of the yolk sac, which often occurs a t 14 to 15 days. Leakage of contents from ruptured yolks occasionally cont inues to such an ex ten t that by 17 days (Fig. 15) the surface area of the CAM is significantly reduced. Reduct ion of CAM sur­face area may result in decreased embryonic respiratory capacity and death of the embryo in such cases. Exposed yolk material is the site of

FIG. 13. View from beneath a culture chamber after 14 days of total incubation. Downgrowth of the extraembryonic membranes leaves only a small por­tion of the yolk (y) uncovered (arrows). The head of the embryo (h) extends beyond the margin of the yolk. Bar equals 1 cm.

FIG. 14. Bottom view of egg contents removed from culture chamber after 17 days of total incuba­tion. Note that the margins of the extraembryonic membranes have fused (lower arrow) around the yolk (y) and embryo (e). The right eye (top arrow) of the embryo is visible. Bar equals 1 cm.

contaminat ion in some cultures. "Compensatory torsion" of Cultured Em­

bryos. Upon transfer of embryos and egg contents to culture chambers after three days of incubat ion in ovo, it has been observed that a small percentage of developing embryos lie with the right side of the cephalic por t ion of their bodies on the yolk rather than the normal

FIG. 15. Yolk sac rupture (r) in egg contents after 17 days of total incubation. Note reduction in CAM surface area. Embryo (e) is visible beneath die CAM.

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376 B. E. DUNN AND M. A. BOONE

left side. By four days torsion is usually com­plete and the entire right side lies on the yolk. At five days (Fig. 16), the sizes of the yolk sac and allantois are similar to the sizes of the respective membranes in normal (left-side) cultured embryos (Fig. 4) . Although expansion of t he allantois occurs at a normal rate in right-side cul tured embryos , the allantois is located dorsally with respect to the embryo rather than ventrally as in left-side cultured embryos (compare Fig. 4, 16). Between five and six days, the dorsal por t ion of the embryo moves u p away from the yolk and the entire embryo ro ta tes 180° to lie entirely upon its left side (Fig. 17). The dorsal por t ion of the embryo now lies to the left of the midbrain, whereas it was to the right of the midbrain at five days (Fig. 16). We have termed such rota t ion from right t o left side "compensa tory tors ion" . A few of the embryos on their right side a t three days do not undergo "compensa­tory tors ion" , bu t instead develop th roughout the period of cul ture on their right side. Embryos remaining on their right side during culture generally survive beyond two weeks of total incubat ion.

The advantages afforded by our cul ture system are as follows: 1) high survival rate, especially during the initial two-thirds of incu­bat ion; 2) rapid downgrowth of ext raembry­onic membranes ; 3) easy observation of cul-

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FIG. 17. Right side cultured embryo after rotation to its left side by six days of total incubation. Arrows are as in Fig. 16. Note that the dark arrow indicating the dorsal portion of the embryo is now on the left side of the midbrain (m). Fig. 16, 17 show the same cultured embryo at different incubation ages. Bar equals 1 cm.

Lured embryos ; and 4) negligible maintenance required to support cultures. The high survival rate can be a t t r ibuted to the large open surface area of the chamber (cf. Corner and Richter,

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FIG. 16. Cultured embryo on right side after five days of total incubation. The midbrain (m) and dorsal portion of the embryo (dark arrow) can be observed. The allantois (clear arrows) lies dorsally with respect to the embryo. Bar equals 1 cm.

FIG. 18. Cultured embryo which failed to undergo "compensatory torsion" shown on its right side after 12 days of total incubation. Labeled are allantoic vessels (a), midbrain (m), left wing (w), left leg (1) and the dorsal portion of the embryo (arrow). Bar equals 1

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CULTURED CHICK EMBRYOS 3 77

1973; Auerbach et al, 1974; Dunn and Boone, 1976) and to the sloping contours of the chamber sides and bottom. Thus the mechani­cal stress imposed by placing the yolk flat on the bottom of a petri dish, which often results in early rupture of the yolk (Auerbach et al., 1974), is alleviated.

The shape of our culture chamber is, how­ever, far from egg-like. Abnormal chamber geometry and lack of turning of culture cham­ber during incubation probably restrict albu­men assimilation in vitro (Dunn and Boone, 1976). As albumen is the major source of embryonic protein during the latter third of incubation (Romanoff, 1967; Freeman and Vince, 1974), it is not surprising that cultured embryos exhibit depressed growth during this period (Dunn and Boone, 1977). Other possible causes of reduced growth and subsequent mor­tality in cultured embryos, such as inadequate calcium supply, excessive humidity, and inhib­ited gas exchange have been suggested (Dunn and Boone, 1976).

Abnormal chamber geometry and lack of turning may also restrict interaction between the yolk and embryo during culture. In ovo there is a tendency for the yolk to extend over the beak, forehead, and legs by 13 to 14 days (Romanoff, I960; Freeman and Vince, 1974). Kuo (1967) has suggested that this movement of the yolk over the ventral surface of the embryo is essential for functional development of the legs and feet by providing a resistance to their extension. The latter author has also indicated that normal yolk orientation is crucial for retraction of the yolk sac prior to hatching. In vitro, however, the embryo remains on top of the yolk throughout the duration of culture.

It is unknown whether the phenomenon of "compensatory torsion" occurs in ovo, or if it is physiologically significant. Heterotaxia, or turning of the early embryo on its right side, is often associated with ectopic viscera in ovo (Romanoff, 1972). However, we have not observed a single case of ectopia in heterotaxic cultured embryos in this system. In ovo, one might expect heterotaxia to be associated with malpositions at the time of hatching, but at least some results indicate that no such associa­tion occurs (Taylor, 1934).

"Pipping" of the CAM with the beak, peeping, and complete yolk sac retraction are

events which occur in ovo immediately prior to hatching. The occurrence of these events in culture suggests that a few embryos have developed almost to the point of hatching outside of the shell. Further refinements in the technique may soon allow successful hatching of cultured embryos.

ACKNOWLEDGEMENTS

The authors express appreciation to Mr. James Martin of the Clemson Communications Center for technical assistance with the photo­graphic equipment.

REFERENCES

Auerbach, R., R. Arensman, L. Kubai, and J. Folk-man, 1975. Tumor-induced angiogenesis: Lack of inh ib i t ion by irradiation. Int. J. Cancer 15:241-245.

Auerbach, R., L. Kubai, D. Knighton, and J. Folkman, 1974. A simple procedure for long-term cultivation of chicken embryos. Devel. Biol. 41:391—394.

Corner, M. A., and A. P. J. Richter, 1973. Extended survival of the chick embryo in vitro. Experientia 29:467-468.

Dunn, B. E., 1974. Technique for shell-less culture of the 7 2-hour avian embryo. Poultry Sci. 53:409-412.

Dunn, B. E., and M. A. Boone, 1976. Growth of the chick embryo in vitro. Poultry Sci. 55:1067—1071.

Dunn, B. E., and M. A. Boone, 1977. Growth and mineral content of cultured chick embryos. Poul­try Sci. 56:662-672.

Freeman, B. M., and M. A. Vince, 1974. Development of the avian embryo. John Wiley and Sons, New York.

Hamilton, H. L., 1952. Lillie's development of the chick. Holt Rinehart and Winston, New York.

Klagsbrun, M., D. Knighton, and J. Folkman, 1976. Tumor angiogenesis activity in cells grown in tissue culture. Cancer Res. 36:110—114.

Kuo, Z. Y., 1967. The dynamics of behavior develop­ment. Random House, New York.

Patten, B. M., 1971. Early embryology of the chick. 5th ed. McGraw-Hill, New York.

Rol'nik, V. V., 1968. Bird embryology. Transl. from Russian by Israel Program for Scientific Transla­tions, 1970. Keter Press, Jerusalem.

Romanoff, A. L., 1960. The avian embryo. Macmillan, New York.

Romanoff, A. L., 1967. Biochemistry of the avian embryo. John Wiley and Sons, New York.

Romanoff, A. L., 1972. Pathogenesis of the avian embryo. Wiley-Interscience, New York.

Taylor, L. W., 1934. Heterotaxia in chick embryos. Poultry Sci. 13:378.

Vogelaar, J. P. W., and J. B. van den Boogert, 1925. Development of the egg of Gallus Domesticus in vitro. Anat. Rec. 30:385—395.

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