A method has been devised to study the nutritional role of yolk cholesterol in the development of the chicken (Gallus gallus domesticus) embryo using egg yolk replacement. Preliminary experiments revealed that 11 mL of yolk in medium-sized eggs were adequate to support embryos from 4 to 20 days of incubation, and that low levels of β-cyclodextrin (β-CD) and amyloglucosidase (AG) in yolk were tolerated by the embryo. Fresh egg yolk was treated with β-CD at 50 °C, centrifuged to remove the insoluble β-CD-cholesterol complex, and incubated with AG to degrade residual β-CD. The cholesterol content was reduced from 13.1 mg g−1 in fresh egg yolk to 1.3 mg g−1 in cholesterol-reduced yolk supernatant (CRYS). Yolk granules co-precipitated with the β-CD-cholesterol complex. The cholesterol concentration of CRYS supplemented with granules from fresh yolk (i.e. CRYSg) was 3.2 mg g−1. When yolks of early 5-day embryos were replaced with 11 mL of CRYS, the maximum survival was 17 days of incubation. When yolks were replaced with 11 mL CRYSg, 30% of embryos survived to 20 days, a significant increase in survival compared to CRYS (P < 0.05). Sixty per cent of embryos receiving fresh yolk (yolk controls) and 83% of embryos in intact eggs (intact controls) survived to 20 days. The controls did not differ significantly (P > 0.05). Embryos in the CRYS and CRYSg groups had shorter survivals than yolk controls (P < 0.001) and intact controls (P < 0.005). These results suggest a methodological approach for investigations of egg yolk cholesterol and yolk granules in chick embryo development.
AbellL.L., LevyB.B., BrodieB.B., and KendallF.E. (1952) A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. Biol. Chem., 195, 357–366.
2.
AntinP.B., FallonJ.F., and SchoenwolfG.C. (2004) The chick embryo rules (still)!Dev. Dyn., 229, 413.
3.
AusticR.E., GrauC.R., and MattesonG.C. (1966) Requirements for glucose and amino acids in defined media for chick embryos. J. Nutr., 90, 175–182.
4.
AusticR.E., and GrauC.R. (1971) Degeneration of the eyes and central nervous system of chick embryos subjected to phenylalanine or tyrosine deficiency. Dev. Biol., 25, 159–173.
5.
AusticR.E., HillF.W., and WilsonB.W. (1971) Role of yolk in chick embryo mortality induced by the feeding of Sterculia foetida oil. Poult. Sci., 50, 1320–1326.
6.
AwadA.C., BenninkM.R., and SmithD.M. (1997) Composition and functional properties of cholesterol reduced egg yolk. Poult. Sci., 76, 649–653.
7.
BasappaC., RaoP., RaoD.N., and DivakarS. (1998) A modified colorimetric method for the estimation of β-cyclodextrin using phenolphthalein. Int. J. Food Sci. Technol., 33, 517–520.
8.
BauerR., PlieschnigJ.A., FinkesT., RieglerB., HermannM., SchneiderW.J. (2013) The developing chicken yolk sac acquires nutrient transport competence by orchestrated differentiation process of its endodermal epithelial cells. J. Biol. Chem., 288, 1088–1098.
9.
BeyerR.S., and JensenL.S. (1989) Overestimation of the cholesterol content of eggs. J. Agric. Food Chem., 37, 917–920.
10.
BurleyR.W., and VadehraD.V. (1989) The avian egg: chemistry and biology, pp. 189–192. John Wiley & Sons, New York.
11.
CooperM.K., WassifC.A., KrakowiakP.A., TaipaleJ., GongR., KelleyR.I., PorterF.D., and BeachyP.A. (2003) A defective response to hedgehog signaling in disorders of cholesterol biosynthesis. Nat. Genet., 33, 508–513.
12.
CotterillO.J., MarionW.W., and NaberE.C. (1977) A nutrient re-evaluation of shell eggs. Poult. Sci., 56, 1927–1934.
13.
DaveyM.G., and TickleC. (2007) The chicken as a model for embryonic development. Cytogenet. Genome Res., 117, 231–239.
14.
ElkinR.G., YanZ., ZhongY., DonkinS.S., BuhmanK.K., StoryJ.A., TurekJ.J., PorterR.E.Jr., AndersonM., HomanR., and NewtonR.S. (1999) Select 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors vary in their ability to reduce egg yolk cholesterol levels in laying hens through alteration of hepatic cholesterol biosynthesis and plasma VLDL composition. J. Nutr., 129, 110–119.
15.
ElkinR.G., and YanZ. (1999) Relationship between inhibition of mevalonate biosynthesis and reduced fertility in laying hens. J. Repro. Fertil., 116, 269–275.
16.
ElkinR.G., FurumotoE.J., and ThomasC.R. (2003) Assessment of egg nutrient compositional changes and residue in eggs, tissues, and excreta following oral administration of atorvastatin to laying hens. J. Agric. Food Chem., 51, 3473–3481.
17.
EtchesR.J. (1996) Reproduction in poultry, pp. 21–23. CAB International, Wallingford.
18.
GoelM.K., KhannaP., and KishoreJ. (2010) Understanding survival analysis: Kaplan–Meier estimate. Int. J. Ayurveda Res., 1, 274–278.
19.
GrauC.R., KleinN.W., and LauT.L. (1957) Total replacement of the yolk of chick embryos. J. Embryol. experiment Morph., 5, 210–214.
20.
GrauC.R., FritzH.I., WalkerN.E., and KleinN.W. (1962) Nutrition studies of chick embryos deprived of yolk. J. Exp. Zool., 150, 185–194.
21.
GrauC.R., WalkerN.E., FritzH.I., and PetersS.M. (1963) Successful development of chick embryos nourished by yolk-sac perfusion with calcium-low medium. Nature, 197, 257–259.
22.
GrauC.R., AusticR.E., and MattesonG.C. (1965) Degeneration of the eyes of tyrosine-deficient chick embryos. Science, 148, 1743–1745.
GreengardO.A., SentenacA., and MendelsohnN. (1964) Phosvitin, the iron carrier of egg yolk. Biochim. Biophys. Acta, 90, 406–407.
25.
HermannM., MahonM.G., LindstedtK.A., NimpfJ., and SchneiderW.J. (2000) Lipoprotein receptors in extraembryonic tissues of the chicken. J. Biol. Chem., 275, 16837–16844.
26.
HimmelsteinD.S., BiC., ClarkB.S., BaiB., and KohtzJ.D. (2010) Balanced Shh signaling is required for proper formation and maintenance of dorsal telencephalic midline structures. BMC Develop. Biol., 10, 118–129.
27.
KleinN.W., GrauC.R., and GreenN.J. (1958) Yolk-sac perfusion of chick embryos: effects of various media on survival. Proc. Soc. Exp. Biol. Med., 97, 425–428.
28.
KainK.H., MillerJ.W.I., Jones-ParisC.R., ThomasonR.T., LewisJ.D., BaderD.M., BarnettJ.V., and ZijlstraA. (2014) The chick embryo as an expanding experimental model for cancer and cardiovascular research. Dev. Dyn., 243, 216–228.
KleinN.W., McConnellE., and Riquier (1964) Enhanced growth and survival of explanted chick embryos cultured under high levels of oxygen. Dev. Biol., 10, 17–44.
31.
KurisakiJ., YamauchiK., IsshikiJ., and OgiwaraS. (1981) Differences between α- and β-lipovitellin from hen egg yolk. Agric. Biol. Chem., 45, 699–704.
32.
LowryO.H., RosebroughN.J., FarrA.L., and RandallR.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275.
33.
MarcoC., AlejandreM.J., Gonzalez-PacanowskaD., SegoviaJ.L., and Garcia-PeregrinE. (1985) Evolution of 3-hydroxy-3-methylglutaryl-CoA reductase and microsomal membrane fluidity throughout chick embryo development. Int. J. Biochem., 17, 271–274.
34.
MorckT.A., and AusticR.E. (1981) Iron requirements of white leghorn hens. Poult. Sci., 60, 1497–1503.
35.
MorckT.A. (1978) Studies on the iron requirement of laying and breeding hens. PhD thesis, Cornell University, Ithaca, NY.
36.
NaberE.C. (1979) The effect of nutrition on the composition of eggs. Poult. Sci., 58, 518–528.
37.
PorterJ.A., YoungK.E., and BeachyP.A. (1996) Cholesterol modification of hedgehog signaling proteins in animal development. Science, 274, 255–259.
38.
PorterF.D. (2002) Malformation syndromes due to inborn errors of cholesterol synthesis. J. Clin. Invest., 110, 715–724.
39.
RaoP., KumarS.U., RaoD.N., and DivakarS. (2000) Optimization of digestion parameters for the elimination of residual β-cyclodextrin used for cholesterol reduction in egg using glucoamylase. Eur. Food Res. Technol., 210, 231–236.
40.
ReisA.H., Almeida-CoburnK.L., LouzaM.P., CerqueiraD.M., AguiarD.P., Silva-CardosoL., MendesF.A., AndradeL.R., Einicker-LamasM., AtellaG.C., BritoJ.M., and AbreuJ.G. (2012) Plasma membrane cholesterol depletion disrupts prechordal plate and affects early forebrain patterning. Dev. Biol., 365, 350–362.
41.
RichardsM.P. (1997) Trace mineral metabolism in the avian embryo. Poult. Sci., 76, 152–164.
42.
RoddaD.D., FriarsG.W., GavoraJ.S., and MerrittE.S. (1977) Genetic parameter estimates and strain comparisons of egg compositional traits. Br. Poult. Sci., 18, 459–473.
43.
RomanoffA.L. (1960) The avian embryo, pp. 1077–1078. The Macmillan Company, New York.
44.
RomanoffA.L. (1967) Biochemistry of the avian embryo, pp. 250–254. Interscience Publishers, John Wiley and Sons, New York.
45.
SaitoZ., MartinW.G., and CookW.H. (1965) Changes in major macromolecular fractions of egg yolk during embryogenesis. Can. J. Biochem., 43, 1755–1770.
SmithD.M., AwadA.C., BenninkM.R., and GillJ.L. (1995) Cholesterol reduction in liquid egg yolk using β-cyclodextrin. J. Food Sci., 60, 691–720.
48.
SprattN.T.Jr., (1949) Nutritional requirements of the early chick embryo. I. The utilisation of carbohydrate substrates. J. Exp. Zool., 110, 273–298.
49.
USDA: (1969) Egg grading manual. Agriculture Handbook No. 75, U.S. Department of Agriculture, Consumer and Marketing Service, Washington D.C.
50.
VarjasaloM., and TaipaleJ. (2008) Hedgehog: functions and mechanisms. Genes Dev., 22, 2454–2472.
51.
WalkerN.E., and GrauC.R. (1963) Growth and development of chick embryos supplied with various concentrations of yolk. J. Nutr., 79, 205–210.
52.
WalkerN.E. (1967) Growth and development of chick embryos nourished by fractions of yolk. J. Nutr., 92, 111–117.
53.
WallaceR.A., and MorganJ.P. (1986) Isolation of phosvitin: retention of small molecular weight species and staining characteristics on electrophoretic gels. Anal. Biochem., 157, 256–261.
54.
WashburnK.W., and NixD.F. (1974) A rapid technique for extraction of yolk cholesterol. Poult. Sci., 53, 1118–1122.
