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Ring Structure of Avian Egg Yolk1
1418
M. G. MURILLO AND L. S. JENSEN
Potter, L. M., 1973. Using proper ingredients properly for poultry. Proc. Virginia Feed Convention and Nutrition Conference, p. 69-76.
Waibel, P. E., 1968. Amino acids and protein for growing turkeys. Proc. 29th Minnesota Nutrition Conference, p. 149-155.
Ring Structure of Avian Egg Yolk1 C. R. GRAU
Department of Avian Sciences, University of California, Davis, California 95616 (Received for publication November 28, 1975)
POULTRY SCIENCE 55: 1418-1422, 1976
I
T has been recognized for more than a century that deposition of yolk in birds occurs by addition of material to the periphery of the oocyte within a follicle of the ovary (Thomson, 1859). This is a continuous process whereby a single yolk of a laying hen may increase by approximately two grams each day during the rapid phase of growth before ovulation (Gilbert, 1972). The deposited yolk is not homogeneous, although it often appears so. This report shows that yolk varies in staining properties depending on the time of day it is deposited in the follicle. Thus, rings are produced which correspond to daily yolk formation. The rate of yolk deposition was found by Warren and Conrad (1939) to be relatively constant. Riddle (1911), on the other hand, had theorized that the ring structure of yolk,
1. A preliminary report of this work was presented to the 64th Poultry Science Association Meeting, Pullman, Washington August 4, 1975 (Poultry Sci. 54: 1767).
which is visible in cross section of some hard-boiled eggs, could be accounted for by differences in deposition during daytime feeding and nighttime inanition, causing the formation of rings of yellow and white yolk in which the white yolk was presumably similar to the central primordial yolk. However, Conrad and Warren (1939) found that when the ring color was visible it reflected variation in the dietary intake of carotenoids. Feeding a laying hen, or injecting it daily with intravenous doses of a fat-soluble dye revealed the ring structure in a hardcooked yolk (Warren and Conrad, 1939). In domestic birds it has been found, by daily feeding or injection of dye and counting of rings in sections of cooked yolks, that the rapid period of yolk deposition takes 7-11 days for chickens (Gilbert, 1972); 11-15 days for turkeys (Bacon and Cherms, 1968); and 5-7 days for quail (Cotumix coturnix japonica) (Homma et al., 1965; Bacon and Koontz, 1972). Little is known about the time course of yolk deposition in wild birds. Bissonnette
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
ABSTRACT Differences in the staining properties of yolk deposited during night and day were used to estimate the time required to deposit yolk within the ovarian follicle. Whole eggs were frozen to -10° C. to alter the lipoproteins, the shell was removed, and contents fixed in 4% formalin for 18 hours at 60-70° C. The yolk was cut in half, and one half was put into 6% potassium dichromate for 18 hours at 60-70° C. Slices revealed alternating light and dark grey-green or brown rings. The center (latebra) was always light. From timed dye-feeding experiments it was found that lighter staining rings were deposited at night. Chicken eggs had 7-11 light rings; coturnix quail 4-6; turkeys 10-12; and cackling geese 12. All eggs from a variety of bird species showed ring structures. Staining formalin-fixed slices with acidified ferrocyanide yielded rings of Prussian blue, a test for forms of iron. Variation in the nutrition of the reproducing female, or the effects of acute contacts with environmental contaminants can be localized in discrete rings of yolks without experimental intervention during yolk deposition.
1419
EGG YOLK RING STRUCTURE
ture with a mixture of equal parts of 5% potassium ferrocyanide and 6N HC1, washed with water and observed. Other stains were: 5% sodium sulfide; or 0.5% alizarin for 5 minutes, then rinsed several hours with water. Laying chickens or quail were fed commercial-type control diets or pigment-low semipurified diets. These contained casein supplemented with arginine and glycine, starch, minerals, and vitamins, or a similar diet containing isolated soybean protein supplemented with methionine. Food and water were always available. Birds were housed in rooms with controlled temperature (25° C.) and light (14 hours, beginning at 6 a.m.) In a number of experiments over a period of several months, 8 laying hens and 40 laying quail were given capsules containing 40 mg. and 10 mg., respectively, of a dye such as Sudan III, Sudan IV, or Sudan Black B mixed with 5-10 times the weight of glucose as a carrier. The times of feeding the dye were recorded, thus permitting comparison of time of forming a dichromate-staining yolk ring and the time of day when the dye was given.
METHODS
RESULTS
Unincubated or infertile whole eggs were put into a vacuum chamber for several hours in order to degas them (to avoid entrapped bubbles), and then were frozen at - 2 0 ° , usually overnight. The frozen eggs were put into water for a few seconds, the shells were removed, and the frozen contents were put into 2-3 volumes of 4% formalin and put into an oven at 60°-70° for 16-20 hours. After freeing them from the white and vitelline membrane, the yolks were cut in half, and one half put into 6% potassium dichromate and returned to the oven (60°-70°) for 16-20 hours. Then the yolk was rinsed to remove most of the chromate and 1-2 mm. slices were cut for observation.
When the formalin-fixed eggs were cut in half, light and dark yellow rings were often apparent. Sometimes there were differences in waxiness or graininess, but generally the cut surface was smooth. Eggs from hens fed purified diets appeared almost homogeneous. After subsequent dichromate treatment, the cut surface revealed light and dark grey-green or brown rings; however, differentiation was clearer in slices cut below the surface. The variation in pattern and color among eggs was great, but pairs of light and dark rings were discernible in almostall eggs. Yolk being deposited in several follicles at a particular time generally had similar, but not identical staining properties: each follicle produced a unique pattern. Although differences between birds within a species were marked,
Slices were also cut from the formalin-fixed half, treated for 10 minutes at room tempera-
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
and Zujko (1936) measured follicle sizes of starlings on different dates; however, the data cannot be interpreted in relation to growth of individual eggs. Yolk structure has been difficult to study, primarily because methods of fixation for histological or chemical study have been unsatisfactory. Heat denaturation of yolk produces a soft, granular texture that is impermanent. Formalin and other aqueous fixatives penetrate fresh yolk slowly, and do not harden it. After freezing and thawing, however, yolk is a viscous, sticky gel (Moran, 1925), which is very permeable to formalin or other fixatives. By this preliminary freezing step, a stable fixed preparation was produced which was easily sliced and stained to reveal differences in structure. Potassium dichromate solutions produced a striking effect by fixation after freezing: alternating light and dark gray-green or brown rings became apparent. In yolks in which the rings were distinct, a pair of one light and one dark ring marked each 24-hour period of yolk deposition.
1420
C. R. GRAU
If the dichromate-stained yolk was not kept moist, it became very dark over the entire surface, and light rings were no longer apparent. When formalin-fixed yolk slices were stained with acidic potassium ferrocyanide, rings of Prussian blue were found, often, but not consistently equal to the numbers of rings revealed by dichromate, and sometimes extremely variable in width and darkness of stain. Sodium sulfide had a similar effect, yielding dark and light grey rings. The basis for these stains appeared to be variation in forms of iron in different layers of yolk rather than in total iron contents, which in preliminary studies were not found to be highly variable. Part of the central latebra showed intense blue stain. Similar but less clear blue stains were found in hard-cooked eggs not fixed by formalin. In these last eggs three or four fine rings were revealed by sodium sulfide in the latebra. When capsules containing Sudan III were fed at 9 a.m. or 9 p.m. or at 36 hour intervals and the eggs laid were stained with dichromate, it was observed that darkly staining yolk was deposited before the dye fed at
9 a.m. was deposited, and that the dye fed at 9 p.m. was deposited in a light ring. The time required to digest, absorb, and deposit dye fed by capsule is approximately 4 hours in hens (Gilbert, 1973). In quail it has been reported as early as 85 minutes (Bacon and Koontz, 1971); data from this laboratory indicate that 3 hours is a reasonable working estimate (Dobbs and Wathen, 1975). Eggs from hens kept in individual cages under controlled environmental conditions generally showed less clearly differentiated rings than eggs from hens obtained from a local small-farm flock maintained in outdoor pens. Chicken eggs were found to have 7 to 11 pairs of light and dark rings when stained by dichromate, as shown by the example in Figure 1 A. This variation in numbers of rings is compatible with the observations of Warren and Conrad (1939) and of Gilbert (1972), who based their conclusions on daily dosing of hens with dye. In some eggs, dichromatestained rings were poorly differentiated, making it difficult or impossible to obtain a satisfactory count, Prussian blue staining showing more variation than dichromate staining; however in some eggs the blue rings were strikingly clear. Infertile eggs that had been incubated for 20 days exhibited definite ring structures, thus indicating the stability of yolk structure. In embryonated eggs the structure was no longer apparent after 3 or 4 days of incubation. Coturnix quail showed 4 to 7 rings, usually 5, again generally corresponding to data obtained by the dye method (Bacon and Koontz, 1971), as shown in Figure IB. Rings that stained abnormally dark were deposited during a period of feeding after 20-hour food deprivation. It is possible that part of the dark " d a y " rings resulted from feeding after normal nighttime inanition, but data from experiments on this question are not conclusive. An example of an egg of a wild bird about
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
there were even greater differences between birds of different genera or species. When the dichromate solution was applied to the frozen eggs without formalin fixation, inner ring differentiation was excellent, but the outer 1-3 mm. margin of the yolk was very light. This was found to be an artifact that was not seen when ovarian follicles with their enclosed yolk were frozen and fixed. The artifact was reduced by coating the thawed and drained yolk with a starch gel before dichromate treatment, but was more satisfactorily avoided by prior formalin fixation. Rings in halves of yolks that had been cut while frozen as well as whole yolks were stained by dichromate. Yolks heated above 80° before fixation stained to a yellow-brown color when treated with dichromate, but no rings were apparent.
EGG YOLK RING STRUCTURE
1421
FIG. 1. Cut sections of yolks that had been stained with dichromate after freezing and fixing with formalin. A. Chicken egg. The lightly stained rings were probably deposited between 9 p.m. and 6 a.m. B. Coturnix quail egg. C. Cackling goose (Branta canadensis minima) egg.
By using the techniques of freezing, fixing, and staining, it is possible to identify and sample fractions of egg yolk that were deposited at specific times. When used in conjunction with oviposition time, characteristics and composition of yolk deposited at specific times can be obtained without experimental intervention during egg formation. In captive birds to which dye markers can be given, the timing can be accurate to a few hours. It is possible to fix and stain one quadrant of a yolk, and match the stained rings with unstained or even frozen, unfixed quadrants.
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
which little is known concerning time factors in yolk deposition is shown in Figure 1C, where a dichromate-stained egg of the cackling goose (Branta canadensis minima) is presented: 12 rings can be counted. Other wild birds, now being studied, also show rings. The method presented here depends on the altered properties of yolk lipoproteins that result from freezing and thawing, with subsequent easy penetration by fixations. Formalin, dichromate, and various other watersoluble substances permeate frozen-thawed yolk quickly, but penetrate fresh yolk slowly, if at all. The nature of the dark stain produced by dichromate is unknown, and thus the difference in composition of "day yolk" and ""night yolk" cannot be attributed to a specific substance or structure. When cooked yolk was stained with dichromate the ring structure was not apparent, but acidic ferrocyanide treatment did bring out Prussian blue rings. Yolk that was fixed by formalin after freezing and then hard-cooked was found to exhibit rings after staining with dichromate. These observations suggest that the color produced by dichromate results from a structural rather than a compositional variation in the day and night yolk. If, for example, the dichromate color resulted from reaction with an unsaturated fatty acid, it would not be expected that cooking would prevent development of the dark stain.
1422
C. R. GRAU
Frozen yolk rings can then be dissected out and made available for chemical analysis. These techniques may prove useful in identifying yolk variation in relation to environmental variables, particularly nutrients or potential toxicants.
ACKNOWLEDGEMENTS The author thanks J. Dobbs,T. Roudybush, cackling goose egg was collected
by
T.
Roudybush at the U.S.D.I. Clarence Rhode National Wildlife Refuge, Bethel, Alaska as part of a cooperative research program on egg formation in wild birds. Part of this study was carried out at the Agricultural Research Council's Poultry Research Centre, Edinburgh, U.K. during a sabbatical leave. The hospitality and advice of A. B. Gilbert and the staff of the Centre is gratefully acknowledged.
REFERENCES Bacon, W. L., and F. L. Cherms, 1968. Ovarian follicular growth and maturation in the domestic turkey. Poultry Sci. 47: 1303-1314. Bacon, W. L., andM. Koontz, 1971. Ovarian follicular
NEWS AND NOTES (Continued from page 1393) industry. His latest effort was the publication entitled "Pheasants" which has had world-wide recognition. He was the first Canadian to win the Poultry and Egg National Board Egg Science Award, administered by the Poultry Science Association, for his paper given at the annual meeting in 1972. His knowledge of the Canadian poultry industry is extensive. If called upon for information he either had it, or knew where it could be obtained. His knowledge does not only encompass the poultry industry but all allied industries. He has developed a good working relationship with colleges, universities,
governmental agencies, and the poultry trade within Canada, the United States, Australia and several European countries. He is a member of the Poultry Science Association and the Canadian Institute of Food Science and Technology. ONTARIO NOTES Don Slinger has been appointed Poultry Department Manager, United Cooperatives of Ontario. He received a B.S.A. degree from the University of Toronto
(Continued on page 1463)
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
and J. Wathen for advice and criticism. The
growth and maturation in coturnix quail. Poultry Sci. 50: 233-236. Bissonnette, T. H., and A. J. Zujko, 1936. Normal progressive changes in the ovary of the starling (Sturnis vulgaris) from December to April. Auk, 53: 31-50. Conrad, R. M., and D. C. Warren, 1939. The alternate white and yellow layers of yolk in hens' ova. Poultry Sci. 18: 220-224. Dobbs, J., and J. Wathen, 1975. Unpublished data from this laboratory. Gilbert, A. B., 1972. The activity of the ovary in relation to egg production. In: Egg Formation and Production, Eds. B. M. Freeman and P. E. Lake, British Poultry Science, Ltd., Edinburgh, 1972, pp. 3-21. Gilbert, A. B., 1973. Personal communication. Homma, K., W. O. Wilson and L. Z. McFarland, 1965. Yolk dye deposition as an index of ovum maturation in Coturnix. Amer. Zoologist, 5: 194. Moran, T., 1925. The effect of low temperature on hens' eggs. Proc. Roy. Soc. 98(B): 436-456. Riddle, O., 1911. On the formation, significance and chemistry of the white and yellow yolk of ova. J. Morphol. 22: 455-491. Thomson, A., 1859. In: Cyclopedia of Anatomy and Physiology, (Ed. R. B. Todd) Vol. 5 (suppl.) pp. 1-142. Gilbert and Piper, London. (Quoted from Gilbert, A. B., The egg: its physical and chemical aspects, In: Physiology and Biochemistry of the domestic Fowl, Eds. D. J. Bell and B. M. Freeman, 1971. Vol. 3, chapter 58, pp. 1379-1399, Academic Press, London. Warren, D. C , and R. M. Conrad, 1939. Growth of the hen's ovum. J. Agr. Res. 58: 875-893.
M. G. MURILLO AND L. S. JENSEN
Potter, L. M., 1973. Using proper ingredients properly for poultry. Proc. Virginia Feed Convention and Nutrition Conference, p. 69-76.
Waibel, P. E., 1968. Amino acids and protein for growing turkeys. Proc. 29th Minnesota Nutrition Conference, p. 149-155.
Ring Structure of Avian Egg Yolk1 C. R. GRAU
Department of Avian Sciences, University of California, Davis, California 95616 (Received for publication November 28, 1975)
POULTRY SCIENCE 55: 1418-1422, 1976
I
T has been recognized for more than a century that deposition of yolk in birds occurs by addition of material to the periphery of the oocyte within a follicle of the ovary (Thomson, 1859). This is a continuous process whereby a single yolk of a laying hen may increase by approximately two grams each day during the rapid phase of growth before ovulation (Gilbert, 1972). The deposited yolk is not homogeneous, although it often appears so. This report shows that yolk varies in staining properties depending on the time of day it is deposited in the follicle. Thus, rings are produced which correspond to daily yolk formation. The rate of yolk deposition was found by Warren and Conrad (1939) to be relatively constant. Riddle (1911), on the other hand, had theorized that the ring structure of yolk,
1. A preliminary report of this work was presented to the 64th Poultry Science Association Meeting, Pullman, Washington August 4, 1975 (Poultry Sci. 54: 1767).
which is visible in cross section of some hard-boiled eggs, could be accounted for by differences in deposition during daytime feeding and nighttime inanition, causing the formation of rings of yellow and white yolk in which the white yolk was presumably similar to the central primordial yolk. However, Conrad and Warren (1939) found that when the ring color was visible it reflected variation in the dietary intake of carotenoids. Feeding a laying hen, or injecting it daily with intravenous doses of a fat-soluble dye revealed the ring structure in a hardcooked yolk (Warren and Conrad, 1939). In domestic birds it has been found, by daily feeding or injection of dye and counting of rings in sections of cooked yolks, that the rapid period of yolk deposition takes 7-11 days for chickens (Gilbert, 1972); 11-15 days for turkeys (Bacon and Cherms, 1968); and 5-7 days for quail (Cotumix coturnix japonica) (Homma et al., 1965; Bacon and Koontz, 1972). Little is known about the time course of yolk deposition in wild birds. Bissonnette
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
ABSTRACT Differences in the staining properties of yolk deposited during night and day were used to estimate the time required to deposit yolk within the ovarian follicle. Whole eggs were frozen to -10° C. to alter the lipoproteins, the shell was removed, and contents fixed in 4% formalin for 18 hours at 60-70° C. The yolk was cut in half, and one half was put into 6% potassium dichromate for 18 hours at 60-70° C. Slices revealed alternating light and dark grey-green or brown rings. The center (latebra) was always light. From timed dye-feeding experiments it was found that lighter staining rings were deposited at night. Chicken eggs had 7-11 light rings; coturnix quail 4-6; turkeys 10-12; and cackling geese 12. All eggs from a variety of bird species showed ring structures. Staining formalin-fixed slices with acidified ferrocyanide yielded rings of Prussian blue, a test for forms of iron. Variation in the nutrition of the reproducing female, or the effects of acute contacts with environmental contaminants can be localized in discrete rings of yolks without experimental intervention during yolk deposition.
1419
EGG YOLK RING STRUCTURE
ture with a mixture of equal parts of 5% potassium ferrocyanide and 6N HC1, washed with water and observed. Other stains were: 5% sodium sulfide; or 0.5% alizarin for 5 minutes, then rinsed several hours with water. Laying chickens or quail were fed commercial-type control diets or pigment-low semipurified diets. These contained casein supplemented with arginine and glycine, starch, minerals, and vitamins, or a similar diet containing isolated soybean protein supplemented with methionine. Food and water were always available. Birds were housed in rooms with controlled temperature (25° C.) and light (14 hours, beginning at 6 a.m.) In a number of experiments over a period of several months, 8 laying hens and 40 laying quail were given capsules containing 40 mg. and 10 mg., respectively, of a dye such as Sudan III, Sudan IV, or Sudan Black B mixed with 5-10 times the weight of glucose as a carrier. The times of feeding the dye were recorded, thus permitting comparison of time of forming a dichromate-staining yolk ring and the time of day when the dye was given.
METHODS
RESULTS
Unincubated or infertile whole eggs were put into a vacuum chamber for several hours in order to degas them (to avoid entrapped bubbles), and then were frozen at - 2 0 ° , usually overnight. The frozen eggs were put into water for a few seconds, the shells were removed, and the frozen contents were put into 2-3 volumes of 4% formalin and put into an oven at 60°-70° for 16-20 hours. After freeing them from the white and vitelline membrane, the yolks were cut in half, and one half put into 6% potassium dichromate and returned to the oven (60°-70°) for 16-20 hours. Then the yolk was rinsed to remove most of the chromate and 1-2 mm. slices were cut for observation.
When the formalin-fixed eggs were cut in half, light and dark yellow rings were often apparent. Sometimes there were differences in waxiness or graininess, but generally the cut surface was smooth. Eggs from hens fed purified diets appeared almost homogeneous. After subsequent dichromate treatment, the cut surface revealed light and dark grey-green or brown rings; however, differentiation was clearer in slices cut below the surface. The variation in pattern and color among eggs was great, but pairs of light and dark rings were discernible in almostall eggs. Yolk being deposited in several follicles at a particular time generally had similar, but not identical staining properties: each follicle produced a unique pattern. Although differences between birds within a species were marked,
Slices were also cut from the formalin-fixed half, treated for 10 minutes at room tempera-
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
and Zujko (1936) measured follicle sizes of starlings on different dates; however, the data cannot be interpreted in relation to growth of individual eggs. Yolk structure has been difficult to study, primarily because methods of fixation for histological or chemical study have been unsatisfactory. Heat denaturation of yolk produces a soft, granular texture that is impermanent. Formalin and other aqueous fixatives penetrate fresh yolk slowly, and do not harden it. After freezing and thawing, however, yolk is a viscous, sticky gel (Moran, 1925), which is very permeable to formalin or other fixatives. By this preliminary freezing step, a stable fixed preparation was produced which was easily sliced and stained to reveal differences in structure. Potassium dichromate solutions produced a striking effect by fixation after freezing: alternating light and dark gray-green or brown rings became apparent. In yolks in which the rings were distinct, a pair of one light and one dark ring marked each 24-hour period of yolk deposition.
1420
C. R. GRAU
If the dichromate-stained yolk was not kept moist, it became very dark over the entire surface, and light rings were no longer apparent. When formalin-fixed yolk slices were stained with acidic potassium ferrocyanide, rings of Prussian blue were found, often, but not consistently equal to the numbers of rings revealed by dichromate, and sometimes extremely variable in width and darkness of stain. Sodium sulfide had a similar effect, yielding dark and light grey rings. The basis for these stains appeared to be variation in forms of iron in different layers of yolk rather than in total iron contents, which in preliminary studies were not found to be highly variable. Part of the central latebra showed intense blue stain. Similar but less clear blue stains were found in hard-cooked eggs not fixed by formalin. In these last eggs three or four fine rings were revealed by sodium sulfide in the latebra. When capsules containing Sudan III were fed at 9 a.m. or 9 p.m. or at 36 hour intervals and the eggs laid were stained with dichromate, it was observed that darkly staining yolk was deposited before the dye fed at
9 a.m. was deposited, and that the dye fed at 9 p.m. was deposited in a light ring. The time required to digest, absorb, and deposit dye fed by capsule is approximately 4 hours in hens (Gilbert, 1973). In quail it has been reported as early as 85 minutes (Bacon and Koontz, 1971); data from this laboratory indicate that 3 hours is a reasonable working estimate (Dobbs and Wathen, 1975). Eggs from hens kept in individual cages under controlled environmental conditions generally showed less clearly differentiated rings than eggs from hens obtained from a local small-farm flock maintained in outdoor pens. Chicken eggs were found to have 7 to 11 pairs of light and dark rings when stained by dichromate, as shown by the example in Figure 1 A. This variation in numbers of rings is compatible with the observations of Warren and Conrad (1939) and of Gilbert (1972), who based their conclusions on daily dosing of hens with dye. In some eggs, dichromatestained rings were poorly differentiated, making it difficult or impossible to obtain a satisfactory count, Prussian blue staining showing more variation than dichromate staining; however in some eggs the blue rings were strikingly clear. Infertile eggs that had been incubated for 20 days exhibited definite ring structures, thus indicating the stability of yolk structure. In embryonated eggs the structure was no longer apparent after 3 or 4 days of incubation. Coturnix quail showed 4 to 7 rings, usually 5, again generally corresponding to data obtained by the dye method (Bacon and Koontz, 1971), as shown in Figure IB. Rings that stained abnormally dark were deposited during a period of feeding after 20-hour food deprivation. It is possible that part of the dark " d a y " rings resulted from feeding after normal nighttime inanition, but data from experiments on this question are not conclusive. An example of an egg of a wild bird about
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
there were even greater differences between birds of different genera or species. When the dichromate solution was applied to the frozen eggs without formalin fixation, inner ring differentiation was excellent, but the outer 1-3 mm. margin of the yolk was very light. This was found to be an artifact that was not seen when ovarian follicles with their enclosed yolk were frozen and fixed. The artifact was reduced by coating the thawed and drained yolk with a starch gel before dichromate treatment, but was more satisfactorily avoided by prior formalin fixation. Rings in halves of yolks that had been cut while frozen as well as whole yolks were stained by dichromate. Yolks heated above 80° before fixation stained to a yellow-brown color when treated with dichromate, but no rings were apparent.
EGG YOLK RING STRUCTURE
1421
FIG. 1. Cut sections of yolks that had been stained with dichromate after freezing and fixing with formalin. A. Chicken egg. The lightly stained rings were probably deposited between 9 p.m. and 6 a.m. B. Coturnix quail egg. C. Cackling goose (Branta canadensis minima) egg.
By using the techniques of freezing, fixing, and staining, it is possible to identify and sample fractions of egg yolk that were deposited at specific times. When used in conjunction with oviposition time, characteristics and composition of yolk deposited at specific times can be obtained without experimental intervention during egg formation. In captive birds to which dye markers can be given, the timing can be accurate to a few hours. It is possible to fix and stain one quadrant of a yolk, and match the stained rings with unstained or even frozen, unfixed quadrants.
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
which little is known concerning time factors in yolk deposition is shown in Figure 1C, where a dichromate-stained egg of the cackling goose (Branta canadensis minima) is presented: 12 rings can be counted. Other wild birds, now being studied, also show rings. The method presented here depends on the altered properties of yolk lipoproteins that result from freezing and thawing, with subsequent easy penetration by fixations. Formalin, dichromate, and various other watersoluble substances permeate frozen-thawed yolk quickly, but penetrate fresh yolk slowly, if at all. The nature of the dark stain produced by dichromate is unknown, and thus the difference in composition of "day yolk" and ""night yolk" cannot be attributed to a specific substance or structure. When cooked yolk was stained with dichromate the ring structure was not apparent, but acidic ferrocyanide treatment did bring out Prussian blue rings. Yolk that was fixed by formalin after freezing and then hard-cooked was found to exhibit rings after staining with dichromate. These observations suggest that the color produced by dichromate results from a structural rather than a compositional variation in the day and night yolk. If, for example, the dichromate color resulted from reaction with an unsaturated fatty acid, it would not be expected that cooking would prevent development of the dark stain.
1422
C. R. GRAU
Frozen yolk rings can then be dissected out and made available for chemical analysis. These techniques may prove useful in identifying yolk variation in relation to environmental variables, particularly nutrients or potential toxicants.
ACKNOWLEDGEMENTS The author thanks J. Dobbs,T. Roudybush, cackling goose egg was collected
by
T.
Roudybush at the U.S.D.I. Clarence Rhode National Wildlife Refuge, Bethel, Alaska as part of a cooperative research program on egg formation in wild birds. Part of this study was carried out at the Agricultural Research Council's Poultry Research Centre, Edinburgh, U.K. during a sabbatical leave. The hospitality and advice of A. B. Gilbert and the staff of the Centre is gratefully acknowledged.
REFERENCES Bacon, W. L., and F. L. Cherms, 1968. Ovarian follicular growth and maturation in the domestic turkey. Poultry Sci. 47: 1303-1314. Bacon, W. L., andM. Koontz, 1971. Ovarian follicular
NEWS AND NOTES (Continued from page 1393) industry. His latest effort was the publication entitled "Pheasants" which has had world-wide recognition. He was the first Canadian to win the Poultry and Egg National Board Egg Science Award, administered by the Poultry Science Association, for his paper given at the annual meeting in 1972. His knowledge of the Canadian poultry industry is extensive. If called upon for information he either had it, or knew where it could be obtained. His knowledge does not only encompass the poultry industry but all allied industries. He has developed a good working relationship with colleges, universities,
governmental agencies, and the poultry trade within Canada, the United States, Australia and several European countries. He is a member of the Poultry Science Association and the Canadian Institute of Food Science and Technology. ONTARIO NOTES Don Slinger has been appointed Poultry Department Manager, United Cooperatives of Ontario. He received a B.S.A. degree from the University of Toronto
(Continued on page 1463)
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on April 1, 2015
and J. Wathen for advice and criticism. The
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