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Ultrasonography as a Tool for Monitoring In Ovo Chicken Development.
Ultrasonography as a Tool for Monitoring In Ovo Chicken Development. 1. Technique and Morphological Findings1-2 CHARLES R. PUGH> 4 E. DAVID PEEBLES, NANCY P. PUGH, and MICKEY A. LATOUR Department of Clinical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6010 and
ABSTRACT Preliminary studies were performed to develop a method for using real-time, B-mode ultrasonography (US) to directly image the internal morphology of the chicken egg and developing embryo. Different soft tissue interfaces will reflect US waves differentially. These reflected waves, or echoes are then converted into a two-dimensional image of internal morphology. A major limitation of diagnostic US is its inability to penetrate through gas or hard tissue (bone, shell) interfaces. Methodology development to overcome the acoustic obstacle presented by the eggshell and air cell constituted the initial part of the preliminary study. An acoustical window was achieved by creating a 2-cm fenestration through ihe large end of the eggshell, then filling the air cell with sterile saline. Morphological features of the yolk and embryo were recorded at 0, 2, 6, 9, 14, and 17 days of incubation. The second part of the preliminary study explores whether the acoustic window, once created, could then be closed, and if closed, whether egg viability could be maintained. A second concurrent trial was conducted with 32 eggs that were fenestrated, imaged, recorded, reclosed, and incubated. Two methods of closure were attempted: one using dialysis membrane and tape; the other using an eggshell allograft. Hatchability was partially retained with both window closure methods. {Key words: ultrasound, egg, chicken embryogenesis, incubation, hatchability) 1993 Poultry Science 72:2236-2246
INTRODUCTION Received for publication May 28, 1993. Accepted for publication August 31, 1993. 1 This is Journal Article Number J-8268 from the Mississippi Agricultural and Forestry Experiment Station. 2 Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Extension Service of these products, or similar ones not mentioned. department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762. Present address: Department of Clinical Studies, Section of Radiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. 4 To whom correspondence should be addressed.
Ultrasonography (US) is a potential useful tool for monitoring in ovo avian embryogenesis and the responses of specific tissues and organs to various external stimuli or treatments. To date, there is a lack of experimental work on the application of alternative imaging methods relative to avian embryogenesis. 31 P-Nuclear magnetic resonance imaging has been used to characterize the yolk and albumen of eggs (Burt et al, 1986). Focused US has been used for the selective destruction of specific tissues in the chicken embryo's central nervous system
2236
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
Department of Poultry Science, Mississippi State University, Mississippi State, Mississippi 39762
2237
ULTRASONOGRAPHY OF THE CHICKEN EGG
sterile needle and syringe, the air cell immediately beneath the eggshell was filled with sterile saline, which served as an acoustic coupler. Eggs were scanned with an aseptically prepared 7.5 MHz convex-linear transducer and ALOKA 650 US machine5 (Figure 1). Images were recorded with a multiformat camera and videotape recorder. Morphological features of the yolk and embryo were recorded at 0, 2, 6, 9, 14, and 17 days of incubation. A second concurrent trial was conducted with 32 fertile broiler eggs to develop a method for acoustic window closure. The goal of this experiment was to determine whether hatchability could be retained despite the relatively invasive nature of the procedure. Two methods of closure were attempted: porous dialysis membrane and tape (FM) were used to close the window in half of the test eggs; the second half was closed using an
7.5 MHZ ConvexLinear Transducer
saline filled air cell
MATERIALS AND METHODS
The first step was to establish a technique for creating an acoustic window in the eggshell: entry site for the ultrasound beam free of interfering gas or hard tissues. In the first trial, approximately 20 infertile and fertile broiler eggs at various stages of incubation were used to develop the window methodology. An acoustical window was created by cutting a 2-cm hole through the large end of the eggshell using a needle probe and small iris scissors. The procedure was performed aseptically using betadine solution to wipe the eggshell and instruments. Using a
sCorometrics Medical Systems, Inc., Wallingford, CT 06492-0333.
membrane
ALBUMEN
YOLK
FIGURE 1. Schematic of the ultrasound procedure. The transducer is ultrasonographically coupled by sterile saline to the interior of the chicken egg through an opening created in the eggshell.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
(Karamyan et ah, 1986). No research has been conducted that utilizes US for realtime imaging of the avian egg or embryo during development. Diagnostic US waves in the range of 2 to 10 MHz are generated by piezoelectric crystals housed within a transducer attached to an US machine (Kremkau, 1989). The US wave is generated through the transducer and transmitted into the subject. The transducer then receives returning US waves, which are reflected (or echoed) from interfaces at various depths within the subject. These returning echoes are then processed by the US computer into a two-dimensional image that is displayed on a monitor. The fact that interfaces between different tissue components will differentially reflect US waves has made US a valuable and accepted tool in veterinary medicine and animal science for the noninvasive imaging of internal structures. Practical applications of US include tissue characterization and guided biopsy of diseased organs, early detection of pregnancy, and the grading of meats. The major limitation to diagnostic US is the inability of US waves to penetrate through gas or hard tissue (bone, shell). The eggshell and air cell of the chicken egg, therefore, present a substantial obstacle to US imaging. A series of trials were conducted in an attempt to overcome these obstacles and to develop a method for examining the internal morphological features of live, developing chicken eggs.
2238
PUGH ET Ah.
WHITE (Black
YOLK Rings)
YELLOW YOLK, (Gray Rings)
VITELLIN MEMBRANE
SHELL
FIGURE 2. Morphological features of chicken egg at Day 0 of incubation. Top) In ovo sonogram (1 cm. markings are along the left border of the sonogram). Bottom) Accompanying graphic diagram.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
TRANSDUCER INTERFACE
DAY 0
ULTRASONOGRAPHY OF THE CHICKEN EGG
2239
GERMINAL DISC
-^easxsx&r FIGURE 3. Fertile chicken egg at Day 2 of incubation with developing germinal disc imaged at slightly different planes of orientation. Top) In ovo sonogram. Bottom) Accompanying graphic diagram.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
DAY 2
2240
PUGH ET AL. project. The 600 eggs were divided into six treatment groups, resulting in 100 of the eggs being subjected to US at Days 0, 2, 6, 9,14, or 17 of incubation. The morphological findings from this study are included in these results. The hatchability portion of the project is reported in a companion study (Peebles et al., 1993). RESULTS AND DISCUSSION Different morphological features of the developing chicken embryo were detectable with US at 0, 2, 6, 9, 14, and 17 days of incubation. Ultrasound recordings of morphology were made during the initial pilot and subsequent hatchability phases of the extended experiment. Morphological observations made at Days 15 and 18 during the earlier studies were not appreciably different from those made at Days 14 and 17 during the larger-scale trial and are, therefore, not addressed separately.
FIGURE 4. In ovo sonograms at two different levels of magnification of developing chick embryo at Day 6 of incubation. Concentric yolk rings are no longer distinct.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
eggshell allograft attached with collodion (CP). All 32 eggs were incubated simultaneously. Windows were created in 16 of the eggs at Day 2 of incubation; half were closed with FM and half were closed with CP. These eggs were then returned to the incubator. These eggs were reopened, examined, and viable eggs reclosed and incubated at Days 9 and 15 of incubation. A second group of 10 eggs was first opened and closed at Day 9. These 10 eggs were reopened, examined, and viable eggs reclosed and incubated at Day 15. A third group of six eggs was opened, closed, and reincubated at Day 18 of incubation. No eggs in any of these groups were analyzed with US at the time of initial opening and closure. Nine randomly selected viable eggs were monitored with US prior to subsequent reclosure. Three of the nine eggs were monitored with US twice. The results of the hatchability pilot trial lead to a large-scale (600 eggs) hatchability
ULTRASONOGRAPHY OF THE CHICKEN EGG
2241 08. 30. 9 0 16:38:02
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PWM
nr|iiTi|iiii|nii|i!ir|iii(m!!|niriiiinini|MMfii 1 2 3 4 5 0
FIGURE 5. Morphological features of Day 9 chick. Top) In ovo sonogram. Bottom) Photograph of extracted Day 9 chick.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
¥r-.m art ea
2242
PUGH ET AL.
White and yellow yolk rings, latebra, vitelline membrane, albumen, and shell were clearly imaged in Day 0 eggs (Figure 2). A hyperechoic developing embryo surrounded by a hypoechoic zone was distinctly seen at Day 2 in the center of the fertile egg yolk (Figure 3). Distinct yolk rings, vitelline membrane, and the chalaza within the albumen were also seen at Day 2. At Day 6, the embryonic site was elongated, appeared more complex, internal movement was noted, and rings were no longer distinguishable within the yolk (Figure 4). At Day 9, heartbeat, eyes, limbs, early calcification, and embryonic movements were detected (Figure 5). Calcified skeletal components of the spine and long bones with distal acoustic shadowing were distinct at Day 14 (Figure 6). Eyes and embryonic movement were again seen, and heart rate was measured with the M-mode function of the US machine at Day 14. At Day 17, the eyes, cranial vault, beak, ribs, spine, long bones, and movement were clearly seen (Figures 7 and 8). The cardiac chambers were more distinct, and M-mode and Doppler US were employed to measure heart rate (Figures 8 and 9).
During the hatchability trials, US was one of several stresses imposed upon the eggs. During these trials embryonic death was often obvious by external observation or candling. Evidence of embryonic death was also seen with US. Early embryonic death was detected as cessation in developmental progression with the US features of Day 6 embryo resembling those seen at Day 2 (Figure 10). Lack of embryonic movement or detectable heartbeat could provide US evidence of later embryonic death. In the 32 egg hatchability pilot study, 1 embryo of 16 eggs initially opened at Day 2 hatched and survived. This embryo survived 1 US and 3 CP procedures. A second embryo died during hatch, having become entangled in the closure tape. This embryo underwent 2 US, 2 CP, and 2 FM procedures. Two CP eggs had live embryos at Day 20, but failed to hatch. Two eggs appeared to be infertile. Early embryonic death occurred in three eggs, and late embryonic death occurred in one. One viable egg was intentionally opened at Day 15. Death in five eggs were attributable to physical procedure, i.e., broken
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
FIGURE 6. In ovo sonograms of calcifying spine and long bone in developing chick at Day 14 of incubation.
ULTRASONOGRAPHY OF THE CHICKEN EGG U3:l(?r.i:n _» RNG04 ^ "
2243 0 9 . 07. 90 16:49:38 ID: ESQ 1?
DRY
MINIM. ftULT
t
r?. s ma ca i
2
DAY 17 EYE
CRANIAL; VAULT
BEAK
EYE
FIGURE 7. Cephalic morphology in a developing chick at Day 17 of incubation. Top) In ovo sonograms. Bottom) Accompanying graphic diagram.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
rr. s asa cs puia
2244
PUGH ET AL. 39, 07, 90 16:48:91 IB: ESS 17 B«Y
FIGURE 8. In ovo sonograms of Day 17 developing chick showing calcifying spine and ribs (left image), and close-up image of the heart (right image). Ventricular chambers (VENT), interventricular septum (VS), and site of atrioventricular valve (AW) are labeled on the sonogram.
shells, torn membranes, or breakdown of the FM seal. In the 10 eggs initially opened and closed at Day 9, 7 embryos of 10 hatched and survived (3 CP, 4 FM). Each egg was also reopened and reclosed at Day 15. One embryo was alive at Day 20 but failed to hatch, one egg was broken during CP procedure at Day 15, and one embryo died between Days 9 and 15. In the six eggs initially opened and closed at Day 18, four embryos hatched and survived (2 CP, 2 FM), one died during hatch (FM), and one failed to hatch. Summary analysis of the 32-egg hatchability pilot showed that a total of 56% of the embryos survived to Day 20, 44% attempted to hatch, and 38% hatched and survived. If infertile eggs and those lost to procedural mishap (broken shells, broken membranes, or breakdown of the FM seal) are eliminated from the total, these percentages rise to 75, 58, and 50%, respectively. Seventy-two percent of the eggs that survived to at least Day 20 underwent two or more shell opening and closure procedures. One egg that went to hatch
was opened and closed four times and analyzed by US twice. The CP and FM methods of closure appeared similarly effective, although both chicks that died during hatching were from FM eggs, with the chicks becoming entangled in the tape seal. Eggs initially opened at Day 9 or 15 of incubation had higher survival rates than those opened at Day 2. Of the nine eggs ultrasounded during the reopeningreclosure procedure, six developed to full term, two went to hatch, and three were lost to procedural mishap. Preliminary results indicate that US of eggs may be a potentially useful research tool. Morphological development and growth may be monitored with US. Additionally, the ability to accurately monitor heart rate and rhythm provides at least one potentially useful method to measure metabolic response of the embryo to various external stimuli or treatments. Ultrasound could also potentially be used to guide internal interventional procedures in the egg or embryo. The results of the current study indicate that the method can be employed with a fairly high preservation of chick viability.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
F7. S Q90 C3 PM10 F7. S 890 C3 PH10 EXIT SN. Mil LQ. HN1 TENDON SINNY- SET UP
ULTRASONOGRAPHY OF THE CHICKEN EGG
2245
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
FIGURE 9. In ovo cardiac ultrasonography. Top) B-mode (left image), and M-mode (right image) sonograms of a Day 17 chick. The position of the ventricle is labeled on the B-mode image, and the M-mode tracing of ventricular motion is traced at the corresponding depth on the M-mode image. Bottom) Doppler spectral display from the heart of a Day-18 chick. Rate of heartbeat was calculated directly from M-mode or Doppler tracing.
2246
PUGH ET AL.
REFERENCES Burt, C. T., L. Jeffreys-Smith, and R. E. London, 1986. P-NMR characterization of hen egg yolk and egg white. Biochim. Biophys. Acta 887:118-120. Karamyan, A. L., N. C. Tumanova, and L. A. Polyakova, 1986. Focused ultrasound in experiments studying the development of brain nervous tissue in chick embryogenesis. Translated
from Dokl. Akad. Nauk. SSSR. 284(4):979-982. Kremkau, F. W., 1989. Diagnostic Ultrasound: Principles, Instruments, and Exercises. 3rd ed. W. B. Saunders, Co., Philadelphia, PA. Peebles, E. D., C. R. Pugh, C. R. Boyle, M. A. Latour, N. P. Pugh, and J. D. Brake, 1993. An evaluation of ultrasonography as a potential tool for monitoring chicken embryonic development: 2. Effects of eggshell alteration and ultrasonography on hatchability. Poultry Sd. 72:2247-2253.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
FIGURE 10. Sonographic evidence of early embryonic death in a Day 6, nonviable egg. The sonographic appearance more closely resembles findings in the Day 2 egg (Figure 3) rather than the Day 6 egg (Figure 4), implying that developmental arrest occurred at about Day 2 of incubation.
ABSTRACT Preliminary studies were performed to develop a method for using real-time, B-mode ultrasonography (US) to directly image the internal morphology of the chicken egg and developing embryo. Different soft tissue interfaces will reflect US waves differentially. These reflected waves, or echoes are then converted into a two-dimensional image of internal morphology. A major limitation of diagnostic US is its inability to penetrate through gas or hard tissue (bone, shell) interfaces. Methodology development to overcome the acoustic obstacle presented by the eggshell and air cell constituted the initial part of the preliminary study. An acoustical window was achieved by creating a 2-cm fenestration through ihe large end of the eggshell, then filling the air cell with sterile saline. Morphological features of the yolk and embryo were recorded at 0, 2, 6, 9, 14, and 17 days of incubation. The second part of the preliminary study explores whether the acoustic window, once created, could then be closed, and if closed, whether egg viability could be maintained. A second concurrent trial was conducted with 32 eggs that were fenestrated, imaged, recorded, reclosed, and incubated. Two methods of closure were attempted: one using dialysis membrane and tape; the other using an eggshell allograft. Hatchability was partially retained with both window closure methods. {Key words: ultrasound, egg, chicken embryogenesis, incubation, hatchability) 1993 Poultry Science 72:2236-2246
INTRODUCTION Received for publication May 28, 1993. Accepted for publication August 31, 1993. 1 This is Journal Article Number J-8268 from the Mississippi Agricultural and Forestry Experiment Station. 2 Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Extension Service of these products, or similar ones not mentioned. department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762. Present address: Department of Clinical Studies, Section of Radiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. 4 To whom correspondence should be addressed.
Ultrasonography (US) is a potential useful tool for monitoring in ovo avian embryogenesis and the responses of specific tissues and organs to various external stimuli or treatments. To date, there is a lack of experimental work on the application of alternative imaging methods relative to avian embryogenesis. 31 P-Nuclear magnetic resonance imaging has been used to characterize the yolk and albumen of eggs (Burt et al, 1986). Focused US has been used for the selective destruction of specific tissues in the chicken embryo's central nervous system
2236
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
Department of Poultry Science, Mississippi State University, Mississippi State, Mississippi 39762
2237
ULTRASONOGRAPHY OF THE CHICKEN EGG
sterile needle and syringe, the air cell immediately beneath the eggshell was filled with sterile saline, which served as an acoustic coupler. Eggs were scanned with an aseptically prepared 7.5 MHz convex-linear transducer and ALOKA 650 US machine5 (Figure 1). Images were recorded with a multiformat camera and videotape recorder. Morphological features of the yolk and embryo were recorded at 0, 2, 6, 9, 14, and 17 days of incubation. A second concurrent trial was conducted with 32 fertile broiler eggs to develop a method for acoustic window closure. The goal of this experiment was to determine whether hatchability could be retained despite the relatively invasive nature of the procedure. Two methods of closure were attempted: porous dialysis membrane and tape (FM) were used to close the window in half of the test eggs; the second half was closed using an
7.5 MHZ ConvexLinear Transducer
saline filled air cell
MATERIALS AND METHODS
The first step was to establish a technique for creating an acoustic window in the eggshell: entry site for the ultrasound beam free of interfering gas or hard tissues. In the first trial, approximately 20 infertile and fertile broiler eggs at various stages of incubation were used to develop the window methodology. An acoustical window was created by cutting a 2-cm hole through the large end of the eggshell using a needle probe and small iris scissors. The procedure was performed aseptically using betadine solution to wipe the eggshell and instruments. Using a
sCorometrics Medical Systems, Inc., Wallingford, CT 06492-0333.
membrane
ALBUMEN
YOLK
FIGURE 1. Schematic of the ultrasound procedure. The transducer is ultrasonographically coupled by sterile saline to the interior of the chicken egg through an opening created in the eggshell.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
(Karamyan et ah, 1986). No research has been conducted that utilizes US for realtime imaging of the avian egg or embryo during development. Diagnostic US waves in the range of 2 to 10 MHz are generated by piezoelectric crystals housed within a transducer attached to an US machine (Kremkau, 1989). The US wave is generated through the transducer and transmitted into the subject. The transducer then receives returning US waves, which are reflected (or echoed) from interfaces at various depths within the subject. These returning echoes are then processed by the US computer into a two-dimensional image that is displayed on a monitor. The fact that interfaces between different tissue components will differentially reflect US waves has made US a valuable and accepted tool in veterinary medicine and animal science for the noninvasive imaging of internal structures. Practical applications of US include tissue characterization and guided biopsy of diseased organs, early detection of pregnancy, and the grading of meats. The major limitation to diagnostic US is the inability of US waves to penetrate through gas or hard tissue (bone, shell). The eggshell and air cell of the chicken egg, therefore, present a substantial obstacle to US imaging. A series of trials were conducted in an attempt to overcome these obstacles and to develop a method for examining the internal morphological features of live, developing chicken eggs.
2238
PUGH ET Ah.
WHITE (Black
YOLK Rings)
YELLOW YOLK, (Gray Rings)
VITELLIN MEMBRANE
SHELL
FIGURE 2. Morphological features of chicken egg at Day 0 of incubation. Top) In ovo sonogram (1 cm. markings are along the left border of the sonogram). Bottom) Accompanying graphic diagram.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
TRANSDUCER INTERFACE
DAY 0
ULTRASONOGRAPHY OF THE CHICKEN EGG
2239
GERMINAL DISC
-^easxsx&r FIGURE 3. Fertile chicken egg at Day 2 of incubation with developing germinal disc imaged at slightly different planes of orientation. Top) In ovo sonogram. Bottom) Accompanying graphic diagram.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
DAY 2
2240
PUGH ET AL. project. The 600 eggs were divided into six treatment groups, resulting in 100 of the eggs being subjected to US at Days 0, 2, 6, 9,14, or 17 of incubation. The morphological findings from this study are included in these results. The hatchability portion of the project is reported in a companion study (Peebles et al., 1993). RESULTS AND DISCUSSION Different morphological features of the developing chicken embryo were detectable with US at 0, 2, 6, 9, 14, and 17 days of incubation. Ultrasound recordings of morphology were made during the initial pilot and subsequent hatchability phases of the extended experiment. Morphological observations made at Days 15 and 18 during the earlier studies were not appreciably different from those made at Days 14 and 17 during the larger-scale trial and are, therefore, not addressed separately.
FIGURE 4. In ovo sonograms at two different levels of magnification of developing chick embryo at Day 6 of incubation. Concentric yolk rings are no longer distinct.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
eggshell allograft attached with collodion (CP). All 32 eggs were incubated simultaneously. Windows were created in 16 of the eggs at Day 2 of incubation; half were closed with FM and half were closed with CP. These eggs were then returned to the incubator. These eggs were reopened, examined, and viable eggs reclosed and incubated at Days 9 and 15 of incubation. A second group of 10 eggs was first opened and closed at Day 9. These 10 eggs were reopened, examined, and viable eggs reclosed and incubated at Day 15. A third group of six eggs was opened, closed, and reincubated at Day 18 of incubation. No eggs in any of these groups were analyzed with US at the time of initial opening and closure. Nine randomly selected viable eggs were monitored with US prior to subsequent reclosure. Three of the nine eggs were monitored with US twice. The results of the hatchability pilot trial lead to a large-scale (600 eggs) hatchability
ULTRASONOGRAPHY OF THE CHICKEN EGG
2241 08. 30. 9 0 16:38:02
\imr'-
IMtftKT
PWM
nr|iiTi|iiii|nii|i!ir|iii(m!!|niriiiinini|MMfii 1 2 3 4 5 0
FIGURE 5. Morphological features of Day 9 chick. Top) In ovo sonogram. Bottom) Photograph of extracted Day 9 chick.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
¥r-.m art ea
2242
PUGH ET AL.
White and yellow yolk rings, latebra, vitelline membrane, albumen, and shell were clearly imaged in Day 0 eggs (Figure 2). A hyperechoic developing embryo surrounded by a hypoechoic zone was distinctly seen at Day 2 in the center of the fertile egg yolk (Figure 3). Distinct yolk rings, vitelline membrane, and the chalaza within the albumen were also seen at Day 2. At Day 6, the embryonic site was elongated, appeared more complex, internal movement was noted, and rings were no longer distinguishable within the yolk (Figure 4). At Day 9, heartbeat, eyes, limbs, early calcification, and embryonic movements were detected (Figure 5). Calcified skeletal components of the spine and long bones with distal acoustic shadowing were distinct at Day 14 (Figure 6). Eyes and embryonic movement were again seen, and heart rate was measured with the M-mode function of the US machine at Day 14. At Day 17, the eyes, cranial vault, beak, ribs, spine, long bones, and movement were clearly seen (Figures 7 and 8). The cardiac chambers were more distinct, and M-mode and Doppler US were employed to measure heart rate (Figures 8 and 9).
During the hatchability trials, US was one of several stresses imposed upon the eggs. During these trials embryonic death was often obvious by external observation or candling. Evidence of embryonic death was also seen with US. Early embryonic death was detected as cessation in developmental progression with the US features of Day 6 embryo resembling those seen at Day 2 (Figure 10). Lack of embryonic movement or detectable heartbeat could provide US evidence of later embryonic death. In the 32 egg hatchability pilot study, 1 embryo of 16 eggs initially opened at Day 2 hatched and survived. This embryo survived 1 US and 3 CP procedures. A second embryo died during hatch, having become entangled in the closure tape. This embryo underwent 2 US, 2 CP, and 2 FM procedures. Two CP eggs had live embryos at Day 20, but failed to hatch. Two eggs appeared to be infertile. Early embryonic death occurred in three eggs, and late embryonic death occurred in one. One viable egg was intentionally opened at Day 15. Death in five eggs were attributable to physical procedure, i.e., broken
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
FIGURE 6. In ovo sonograms of calcifying spine and long bone in developing chick at Day 14 of incubation.
ULTRASONOGRAPHY OF THE CHICKEN EGG U3:l(?r.i:n _» RNG04 ^ "
2243 0 9 . 07. 90 16:49:38 ID: ESQ 1?
DRY
MINIM. ftULT
t
r?. s ma ca i
2
DAY 17 EYE
CRANIAL; VAULT
BEAK
EYE
FIGURE 7. Cephalic morphology in a developing chick at Day 17 of incubation. Top) In ovo sonograms. Bottom) Accompanying graphic diagram.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
rr. s asa cs puia
2244
PUGH ET AL. 39, 07, 90 16:48:91 IB: ESS 17 B«Y
FIGURE 8. In ovo sonograms of Day 17 developing chick showing calcifying spine and ribs (left image), and close-up image of the heart (right image). Ventricular chambers (VENT), interventricular septum (VS), and site of atrioventricular valve (AW) are labeled on the sonogram.
shells, torn membranes, or breakdown of the FM seal. In the 10 eggs initially opened and closed at Day 9, 7 embryos of 10 hatched and survived (3 CP, 4 FM). Each egg was also reopened and reclosed at Day 15. One embryo was alive at Day 20 but failed to hatch, one egg was broken during CP procedure at Day 15, and one embryo died between Days 9 and 15. In the six eggs initially opened and closed at Day 18, four embryos hatched and survived (2 CP, 2 FM), one died during hatch (FM), and one failed to hatch. Summary analysis of the 32-egg hatchability pilot showed that a total of 56% of the embryos survived to Day 20, 44% attempted to hatch, and 38% hatched and survived. If infertile eggs and those lost to procedural mishap (broken shells, broken membranes, or breakdown of the FM seal) are eliminated from the total, these percentages rise to 75, 58, and 50%, respectively. Seventy-two percent of the eggs that survived to at least Day 20 underwent two or more shell opening and closure procedures. One egg that went to hatch
was opened and closed four times and analyzed by US twice. The CP and FM methods of closure appeared similarly effective, although both chicks that died during hatching were from FM eggs, with the chicks becoming entangled in the tape seal. Eggs initially opened at Day 9 or 15 of incubation had higher survival rates than those opened at Day 2. Of the nine eggs ultrasounded during the reopeningreclosure procedure, six developed to full term, two went to hatch, and three were lost to procedural mishap. Preliminary results indicate that US of eggs may be a potentially useful research tool. Morphological development and growth may be monitored with US. Additionally, the ability to accurately monitor heart rate and rhythm provides at least one potentially useful method to measure metabolic response of the embryo to various external stimuli or treatments. Ultrasound could also potentially be used to guide internal interventional procedures in the egg or embryo. The results of the current study indicate that the method can be employed with a fairly high preservation of chick viability.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
F7. S Q90 C3 PM10 F7. S 890 C3 PH10 EXIT SN. Mil LQ. HN1 TENDON SINNY- SET UP
ULTRASONOGRAPHY OF THE CHICKEN EGG
2245
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
FIGURE 9. In ovo cardiac ultrasonography. Top) B-mode (left image), and M-mode (right image) sonograms of a Day 17 chick. The position of the ventricle is labeled on the B-mode image, and the M-mode tracing of ventricular motion is traced at the corresponding depth on the M-mode image. Bottom) Doppler spectral display from the heart of a Day-18 chick. Rate of heartbeat was calculated directly from M-mode or Doppler tracing.
2246
PUGH ET AL.
REFERENCES Burt, C. T., L. Jeffreys-Smith, and R. E. London, 1986. P-NMR characterization of hen egg yolk and egg white. Biochim. Biophys. Acta 887:118-120. Karamyan, A. L., N. C. Tumanova, and L. A. Polyakova, 1986. Focused ultrasound in experiments studying the development of brain nervous tissue in chick embryogenesis. Translated
from Dokl. Akad. Nauk. SSSR. 284(4):979-982. Kremkau, F. W., 1989. Diagnostic Ultrasound: Principles, Instruments, and Exercises. 3rd ed. W. B. Saunders, Co., Philadelphia, PA. Peebles, E. D., C. R. Pugh, C. R. Boyle, M. A. Latour, N. P. Pugh, and J. D. Brake, 1993. An evaluation of ultrasonography as a potential tool for monitoring chicken embryonic development: 2. Effects of eggshell alteration and ultrasonography on hatchability. Poultry Sd. 72:2247-2253.
Downloaded from http://ps.oxfordjournals.org/ at Florida Atlantic University on February 26, 2015
FIGURE 10. Sonographic evidence of early embryonic death in a Day 6, nonviable egg. The sonographic appearance more closely resembles findings in the Day 2 egg (Figure 3) rather than the Day 6 egg (Figure 4), implying that developmental arrest occurred at about Day 2 of incubation.