Ultrastructure of Vitelline Membranes from Normal and Mottled Egg Yolks1,2

Ultrastructure of Vitelline Membranes from Normal and Mottled Egg Yolks1 >2 F. E. CUNNINGHAM and D. M. YLANDER Department of Animal Sciences and Industry, Kansas State University, Manhattan, Kansas 66506 (Received for publication November 29, 1979) ABSTRACT This study showed that ultrastruture of the vitelline membrane degenerated with degree of yolk mottling. The more severe the mottling the greater the damage to the membrane. The vitelline membrane appears to be composed of three separate structures. The primary matrix (probably collagen) retains its composition until mottling is most severe; then this structure starts to come apart and lose its integrity. The secondary matrix (probably mucin) aids in holding the primary structure in a fixed position. When this structure is removed there is movement in the primary matrix causing large holes to appear. The tertiary matrix (also mucin) is the quickest to disintegrate. The tertiary matrix covers the other structures much like the cuticle around the egg shell. Once it is removed, the remaining structures are open to stress and damage as mottling increases. (Key words: vitelline membrane, egg yolks, ultrastructure, mottled yolks, electron microscopy) 1980 Poultry Science 59:2449-2 J5 INTRODUCTION

The cause of an abnormality in fresh and stored eggs, referred to as mottled yolks, has been researched for many years. Mottling can occur naturally, but more often it is the result of various feed additives such as cottonseed meal, Nicarbazin (Polin and Porter, 1956), antioxidants, or worming compounds. The motded condition of egg yolks has been described as oily, transparent, orange-yellow spots appearing on the surface of or within the yolk. The mottled yolk is edible but its appearance is often highly objectionable. Most intensely mottled yolks are removed by candling, but occasionally moderately mottled yolks are overlooked by the candler and appear on the consumer's table. Factors commonly associated with mottled yolks were recently reviewed by Cunningham and Sanford (1973, 1974). The coccidiostatic drug, Nicarbazin, has become a research tool because of its ability to produce field-like conditions in the laboratory. The drug must be fed to laying hens at levels of about .005% or higher for at least 4 to 5 days

'Contribution No. 79-64J, Department of Animal Sciences and Industry, Kansas Agricultural Experiment Station, Manhattan, KS 66506. 2 Presented at the 68th Annual Meeting of the Poultry Science Association, University of Florida, Gainesville, FL.

before any significant effect on mottling can be observed. At least 8 days of feeding are required for a maximum effect (Polin, 1960). Nicarbazin fed at levels of .005% and higher increased the severity and incidence of mottling above that of control pens. Levels as high as .03% were fed, and a direct relationship was found between the degree of mottling and the percent Nicarbazin in the diet (Polin and Porter, 1956; Silvestrini et al. 1965a). Baker et al. (1957), Polin et al. (1957), and Silvestrini et al. (1965a,b) reported an increase in mottled yolks in eggs which had been stored at refrigerated temperatures. Baker et al. (1956) and van Tienhoven et al. (1958) reported three general observations about mottled yolks: 1) incidence and severity of mottled yolks in eggs laid by hens fed Nicarbazin increased during storage; 2) mottled areas on yolks produced by hens fed Nicarbazin were associated with movement of water from albumen to the yolk accompanied by a decrease in fat content or an increase in the protein to fat ratio of the yolks; 3) a change in pH of mottled yolks from acid to alkaline accompanied water movement from albumen to yolk. Britton (1973) observed that the protein content of the vitelline membrane decreased with increased degree of mottling; however, there were no gross chemical changes in the vitelline membranes of natural or Nicarbazininduced mottled yolks if the degree of mottling

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CUNNINGHAM AND YLANDER

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TABLE 1. Yolk mottling score card and description of number. Score

10

Degree

Description

None

Normal yolk

Very

Localized spotting, light

Slight

Localized spotting easily detectable, half of yolk involved.

Very moderate

Spotting through entire yolk

Moderate

Yolk becoming orange-brown in color, slight discoloration of thick white

Slightly objectional

Yolk totally orange-brown, thick white same color as yolk

Moderately objectional

Appears to be solids clumped in one part of yolk, light yellow in color

Slightly severe

Slight discoloration of thin white, dark orange yolk, solids cover V> of yolk

Moderately severe

Discoloration of thin white complete, yolk spreads out in thick white

Very severe

Dark brown yolk and thick white, yolk very watery solids orange in color

Complete

Thin white same color as yolk, very little thick white visable, yolk breaks with little resistance

was the same. Yet Nicarbazin tended to increase the rate at which chemical changes occurred compared with that of naturally mottled yolks. Cunningham (1976, 1977) studied the composition of yolks and albumen from eggs laid by hens on rations containing Nicarbazin

and found lower solids and less total lipids in mottled than in nonmottled yolks. Albumen proteins were also present in the mottled yolks, implying migration of lipid material from mottled yolks through the vitelline membrane into the albumen. He suggested that the vitelline

TABLE 2. Egg weights, percent production, and degree of mottling in eggs from hens fed a layer ration with and without Nicarbazin Ave egg

Days on experimental ration

weight Control Nicarb.

10 12 14 16 18 20 22 24 26 28 30

52.3 52.9 52.9 54.8 55.6 53.4 52.9 54.5 55.0 54.9 56.4

52.1 50.6 51.4 47.6 50.6 49.6 51.0 49.4 50.7 48.1 47.2

Percent production Control Nicarb.

Mottling score Control Nicarb.

70 70 78 80 65 70 60 70 80 65 70

1 1 2 1 1 1 3 1 1 1 1

60 40 38 40 50 30 10 20 10 10 5

1 1 2 3 3 4 5 7 8 9 10

ULTRASTRUCTURE OF VITELLINE MEMBRANES

membrane surrounding mottled yolks was more permeable than previously thought. This study was conducted to determine how the ultrastructure of the vitelline membrane changes as die severity of yolk mottling increases. Data on permeability and structural degeneration of die vitelline membrane from motded yolks compared with those of normal yolks are presented.

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MATERIALS AND METHODS

Twenty White Leghorn Hens (Y-strain Kansas State University; 30 weeks old) housed in floor pens were randomly divided into two groups of 10 birds each. One group was fed a standard 18% protein layer ration; the other group was fed the same ration containing .02% (200 ppm) Nicarbazin. The hens had access to feed at all times from hanging feeders.

f

i H a n n oss

5,OOOX

5QOX

g ." *

50-3 ili,0

05 000 BBS

50,OOOX FIG. 1. Electron micrographs of vitelline membranes from normal yolks.

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CUNNINGHAM AND YLANDIiR

Eggs were collected once every 24 hr for a 30-day period from both groups. The eggs were identified by pen, treatment, and date. Daily production per group was recorded. Eggs, selected randomly, were broken out daily and yolks were scored for degree of motding (0 for normal through 10 for those most severely mottled). The remaining eggs were held at 5 C for 30 days, then scored using the same score card (Table 1).

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Once mottling was induced, both mottled and control yolks were rolled on paper towels to remove adhering albumen. Vitelline membranes were separated from the yolk and washed in saline, fixed in gluteraldehyde for 60 min and then placed in an ethanol dilution series (30 to 100%) to replace the gluteraldehyde with ethanol. After removing the samples from ethanol, they were immediately placed in a Denton DCP-1 critical point dryer flushed with

•?;

FIG. 2. Electron micrographs of vitelline membranes from yolks with various degrees of mottling. (5,000 X)

ULTRASTRUCTURE OF VITELLINE MEMBRANES carbon dioxide. Membranes were then m o u n t e d t o aluminum SEM stubs with Pelco No. 93 colloidal silver paint. T h e samples were finally coated with 1 to 2 nm of carbon by vacuum evaporation (Kenny Vacuum Co., Model KSE-2A-M). T h e prepared m e m b r a n e s were examined with an ETEC Autoscan electron microscope operating at 20 Kv accelerating voltage. Pictures of b o t h control and m o t t l e d mem-

- 3 JO.O OS 000 ODH

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branes were taken at various magnifications. Eggs held in storage were b r o k e n o u t after a m i n i m u m of 3 0 days to determine the effects of Nicarbazin during storage. Only eggs from die first 16 days could be used. Those were scored using Table 1. RESULTS T h e effect of Nicarbazin on egg weight, percent production, and m o t t l e d scores is

-3 eo.o os ooi oofi

FIG. 3. Electron micrographs of vitelline membranes from normal (1) and mottled (9) yolks. (25,000 and 50,000 X)

CUNNINGHAM A N D YLANDIiR

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shown in Table 2. Both egg weight and percent production were markedly reduced by 200 ppm of Nicarbazin in the feed. After the 14th day of feeding, all fresh eggs from the hens fed Nicarbazin showed some degree of mottling. During cold storage, all eggs after the 3rd day showed signs of mottling. Egg yolks from hens fed the control ration had a maximum score of 3 after storage, whereas yolks from hens fed the Nicarbazin ration had a maximum mottling score of 10 after storage. Ultrastructure of vitelline membranes from normal yolks is depicted in Figure 1 at 500, 5,000, and 50,000x . The vitelline membrane from normal yolks is seen as consisting of a layer of thick collagen-like fibers and a layer of thinner mucin fibers. These are covered by a thin continuous layer of mucin-like material. Figure 2 shows the ultrastructural disintegration of the vitelline membranes from yolks having progressively greater mottling. Membranes shown were from yolks having mottling scores from 1 to 9. Vitelline membranes from yolks having a mottling score of 10 could not be handled sufficiently for SEM pictures. The contrast between normal and mottled yolk specimens is shown in Figure 3. Vitelline membranes are shown at 25,000 and 50,000x . These figures clearly illustrate the permeability of vitelline membranes from mottled yolks. DISCUSSION

Earlier reports on the vitelline membrane have been inconclusive with respect to physical structure. Liebermann (1888) reported that the vitelline membrane consisted of keratinous material. Lacaillon (1910) found three layers, the middle layer being cellular in structure, whereas the external layers were fibrous. Moran and Hale (1936) also reported three layers and concluded that the two external layers consisted of mucin while the middle layer was keratinous. McNally (1943) found that the vitelline membrane of the freshly ovulated yolk was formed from the collagenous membrane which lines the inner surface of the follicular epithelium. He reported that this membrane became swollen and was covered with a layer of mucin during its passage down the oviduct. Reports on the average thickness of the vitelline membrane range from 15 /J (Lacaillon, 1910) to 47.74 ju (McNally, 1943). Doran and Mueller (1961) reported that the membrane consisted of a layer of collagenous fibers formed in the ovarian follicle and a layer

Primary and secondary matrices

ULTRASTRUCTURE OF VITELLINE MEMBRANES of mucin fibers formed in the magnum. They concluded that the collagen layer was the true vitelline membrane and that the mucin layer corresponded to the chalaziferous membrane. Our SEM study of both normal and mottled yolk membranes indicates that there are three separate layers or matrices present. Those are schematically illustrated in Figure 4. The primary matrix is likely the collagenous layer described by Moran and Hale (1936) and Doran and Mueller (1961). The secondary and tertiary layers appear to be mucin fibers. Our primary and secondary layers are probably the two vitelline membrane layers described by Bellairs et al. (1963), and our tertiary matrix probably coincides widi what Bellairs described as "a very thin, continuous membrane". The SEM photos in Figure 2 illustrate that as mottling becomes more severe, progressive disintegration occurs. First, the tertiary layer breaks up, followed by increasing permeability of the secondary layer. These observations support the suggestion by Feeney et al. (1956) that deterioration of yolk strength occurs under conditions similar to those of the breakdown of uhick albumen. When mottling was severe, the corresponding vitelline membrane was easily torn and very difficult to handle. When mottling was extreme, it was impossible to handle and mount any part of the membrane for SEM study. Our work also agrees with the findings of Baker et al. (1957), Polin et al. (1957), and Silvestrini et al. (1965 a,b) that storage time increases the severity of mottled yolks. It was observed that after the third day of storage 100% of the eggs from hens fed Nicarbazin were mottled. REFERENCES Baker, R. C , F. W. Hill, A. van Tienhoven, and J. H. Bruckner, 1956. Effect of Nicarbazin on egg quality. Poultry Sci. 35:1132. Baker, R. C , F. W. Hill, A. van Tienhoven, and J. H. Bruckner, 1957. The effect of Nicarbazin on egg production and egg quality. Poultry Sci. 36: 718-726. Bellairs, R., M. Harkness, and R. D. Harkness, 1963. The vitelline membrane of the hen's egg: A

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chemical and electron microscopical study. Ultrastructure Res. 8:339-359. Britton, W. M., 1973. Vitelline membrane chemical composition in natural and induced yolk mottling. Poultry Sci. 52:459-464. Cunningham, F. E., 1976. Composition and functional properties of mottled yolks. Poultry Sci. 55: 994-998. Cunningham, F. E., 1977. Properties of albumen from eggs having mottled yolks. Poultry Sci. 56: 1819-1821. Cunningham, F. E., and P. E. Sanford, 1973. Some nutritional aspects of egg yolk mottling. Feedstuffs 45(32):27-30. Cunningham, F. E. and P. E. Sanford, 1974. A review of facts influencing egg yolk mottling. World's Poultry Sci. J. 30:103-114. Doran, B. M., and W. J. Mueller, 1961. The development and structure of the vitelline membrane and their relationship to yolk mottling. Poultry Sci. 40:474-478. Feeney, R. E., J. M. Weaver, J. R. Jones, and M. B. Rhodes, 1956. Studies of the kinetics and mechanisms of yolk deterioration in shell eggs. Poultry Sci. 35:1061-1066. Lacaillon, M. A., 1910. Sur la structure et la significantion de la membrane qui enveloppe la sphere vitelline de l'oeuf des oiseaux. Comptes Rendes Acad. Sci. 150:240-242. Liebermann, L., 1888. Arch F.D. Ges. Physiol. (Pflugers) 43:71. McNally, E. H., 1943. The origin and structure of the vitelline membrane of the domestic fowl's egg. Poultry Sci. 22:40-43. Moran, T., and H. P. Hale, 1936. Physics of the hen's egg. I. Membranes in the egg. J. Exp. Biol. 13:35-40. Polin, D., 1960. Yolk mottling. What causes it? Can you prevent it? Poultry Process. Marketing 66:26, 34, 36. Polin, D., W. H. Ott, and O. H. Siegmond, 1957. The incidence and degree of yolk mottling in eggs from hens fed diets with and without Nicarbazin. Poultry Sci. 36:524-528. Polin, D., and C. C. Porter, 1956. The effect of Nicarbazin on porphyrin and yolk formation. Poultry Sci. 35:1165. Silvestrini, D. A., L. E. Dawson, and R. J. Evans, 1965b. Effects of Nicarbazin in diet on mottled yolks. 2. Lipids. Poultry Sci. 44:1285-1291. Silvestrini, D. A., L. E. Dawson, R. J. Evans, and J. A. Davidson, 1965a. Effects of Nicarbazin in diet on mottled yolks. I. Incidence of degree of mottling, and certain yolk proteins. Poultry Sci. 44:467-473. van Tienhoven, A., F. W. Hill, A. Prock, and R. C. Baker, 1958. The effect of Nicarbazin on yolk quality. Poultry Sci. 37:129-132.