HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, X.-T. Articles by Wu, C.-X. Search for Related Content PubMed PubMed Citation Articles by Wang, X.-T. Articles by Wu, C.-X.

Study of the Deposition Process of Eggshell Pigments Using an Improved Dissolution Method

X.-T. Wang^*,1, X.-M. Deng^*,1, C.-J. Zhao^*, J.-Y. Li^*, G.-Y. Xu^*, L.-S. Lian^*, and C.-X. Wu^*,2

^* State Key Laboratory of Agrobiotechnology and the Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, China Agricultural University, Beijing, 100094, China; and College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China

² Corresponding author: chxwu{at}public.bta.net.cn

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
An improved dissolution method called layer-by-layer dissolution was adopted to study the process of eggshell deposition, which is opposite to the process of eggshell dissolution. In the present study, blue and brown eggshells from 2 Chinese indigenous chicken breeds, Dongxing and Shouguang, respectively, were analyzed with layer-by-layer dissolution. The results showed that the deposition velocity of the eggshell pigments in the top (first) eggshell layer was the highest compared with other layers, which were biliverdin and protoporphyrin in blue eggshell or primarily protoporphyrin in brown eggshell. It was also revealed that the deposition processes of biliverdin and protoporphyrin were synchronous in the blue eggshell of the Dongxiang chicken in the present study.

Key Words: chicken • eggshell color • protoporphyrin • biliverdin

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The eggshell color of wild birds is diverse, whereas the eggshell color of poultry is relatively consistent because of artificial selection. Researchers (Poole, 1965; Kennedy et al., 1976; Ito et al., 1993) discovered that the primary avian eggshell pigments are protoporphyrin IX, biliverdin IX, and biliverdin IX zinc chelate in both wild birds and poultry. The deposition process of brown eggshell pigments has been reported by Baird et al. (1975), but the deposition process of blue eggshell pigments still remains unclear. In most cases, the deposition process of eggshell pigments is studied by collecting uterine fluid. However, collection of uterine fluid cannot avoid surgical stress and other stimulations.

To study the deposition process of eggshell pigments in blue eggshells without stressing the birds, an improved dissolution method called layer-by-layer dissolution was adopted in the present study.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Thirty Dongxiang hens (an indigenous chicken breed in China that lays blue-shelled eggs) and 30 Shouguang hens (an indigenous chicken breed in China that lays brown-shelled eggs) were kept in single cages in 1 poultry house. One egg was collected from each hen and analyzed while fresh. The experimental procedures were as follows. Each egg was cleaned with deionized water, placed in an oven for 3 min until the eggshell surface was dry, and weighed on a microbalance to obtain the initial (NO.0) weight of the egg recorded as W₀. Each egg was placed gently into a 100-mL beaker, into which was poured 45 mL of solvent (methanol:concentrated HCl = 2:1), and kept for 2 min. The egg was removed after 2 min, rinsed in a 5,000-mL beaker with 3,000 mL deionized water, placed in an oven for 3 min until eggshell surface was dry, and weighed on a microbalance to obtain the NO.1 weight of the egg recorded as W₁. Then, 6 mL of solution out of 45 mL of solvent in which the NO.1 eggshell layer was dissolved was transferred into a 10-mL centrifuge tube for analysis and labeled with layer number. The same egg was placed into another clean 100-mL beaker, and procedures 2 and 3 were repeated until the eggshell was dissolved completely. The 6-mL solution for analysis from each layer was placed in a 10-mL centrifuge tube in darkness for 12 h and centrifuged at 1,369.55 x g for 45 min, and the color of the supernatant solution was analyzed in a spectrophotometer at wavelengths of 412 and 670 nm. Absorbance at 412 nm represents the content of protoporphyrin (Ito et al., 1993), and absorbance at 670 nm represents the content of biliverdin (Zhao et al., 2006).

Data analysis was conducted using Microsoft Excel (Microsoft Corporation, Redmond, WA) and SAS 8.2 (SAS Institute, Cary, NC). The calculation methods of parameters were as follows. Weight of each eggshell layer was determined as follows

where n = the layer of eggshell; Wn = weight of the reduced eggshell in the NO.n dissolution, which indicates the weight of the NO.n eggshell layer; Wn-1 = the NO.n–1 weight of the egg; and Wn = the NO.n weight of the egg.

Concentration of protoporphyrin in each eggshell layer was determined as follows

where n = the layer of eggshell; C_pn = concentration of protoporphyrin in the NO.n eggshell layer; A_pn = absorbance of the solution from the NO.n eggshell layer at 412 nm, which indicates the content of protoporphyrin in the NO.n eggshell layer.

Concentration of biliverin in each eggshell layer was determined as follows

where n = the layer of eggshell; C_bn = concentration of biliverdin in the NO.n eggshell layer; A_bn = absorbance of the solution from the NO.n eggshell layer at 670 nm, which indicates the content of biliverdin in the NO.n eggshell layer.

The correlation among A_bn, A_pn, and Wn for the blue eggshells of the Dongxiang chickens was analyzed with the CORR procedure in SAS 8.2.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We found that the deposition patterns of eggshell pigments of 30 blue eggshells were similar as were the 30 brown eggshells. Therefore, the results of 1 blue eggshell and 1 brown eggshell are presented in Figure 1 and Figure 2. With the method of layer-by-layer dissolution, the blue eggshell was divided into 8 eggshell layers, and the brown eggshell was divided into 7 eggshell layers from exterior to interior.

View larger version (9K): [in this window] [in a new window]   Figure 1. Concentrations of protoporphyrin in the layers of eggshell from Dongxiang chickens (layers 1 to 8; ) and the Shouguang chickens (layers 1 to 7; ).

View larger version (10K): [in this window] [in a new window]   Figure 2. Concentrations of biliverdin in the layers of eggshell from Dongxiang chickens (layers 1 to 8; ) and the Shouguang chickens (layers 1 to 7; ).

Figure 2 shows the concentrations of biliverdin in the eggshell layers of the Dongxiang chicken and the Shouguang chicken, respectively. It was found that the concentrations of biliverdin in NO.1 eggshell layer were the highest for both of the 2 color eggshells.

The correlation among A_bn, A_pn, and Wn for the blue eggshells of the Dongxiang chicken are presented in Table 1. The correlation coefficient between A_bn and A_pn was 0.97 (P < 0.01). The correlations between A_bn and Wn or between A_pn and Wn were not significant (P > 0.05). Because of the small quantity of biliverdin, the correlation among A_bn, A_pn, and Wn was not analyzed for the brown eggshells of the Shouguang chicken.

View this table: [in this window] [in a new window]   Table 1. Correlation among Abn, Apn, and Wn for the blue eggshells of the Dongxiang chickens1

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All the eggshell ingredients in each eggshell layer were dissolved except CO₂ released. Therefore, we could study the deposition process of not only the eggshell pigments but also other eggshell ingredients, such as Ca and P, using the layer-by-layer dissolution procedures, which was more convenient compared to other methods. Several points should be emphasized in relation to the experiment: a) the egg must be completely immersed in solvent by adjusting the volume of solvent according to the size of different eggs and b) the division into 8 eggshell layers in the blue eggshell and 7 eggshell layers in the brown eggshell were artificial and not physiological.

The concentrations of protoporphyrin and biliverdin in NO.1 eggshell layer were at the highest level (Figures 1 and 2), which suggested that not only the protoporphyrin but also the biliverdin reached the maximal rate of deposition during the final stage of eggshell formation. Baird et al. (1975) found that most of the brown eggshell pigment (protoporphyrin) was localized in the cuticle of the brown eggshell. However, the distribution of another important eggshell pigment (biliverdin) has not been reported previously. In this study, we found that the distribution of biliverdin throughout the eggshell was similar to that of protoporphyrin, and therefore, it is assumed that the deposition process of biliverdin from shell gland to eggshell was similar to that of protoporphyrin.

The positive correlation between A_bn and A_pn (Table 1; i.e., the deposition velocity of biliverdin fluctuating along with that of protoporphyrin in the deposition process of eggshell) could be interpreted as the synchronization of depositions of biliverdin and protoporphyrin in the process of eggshell formation. The synchronization could be caused by the same stimulus.

Zhao et al. (2006) thought that biliverdin should be synthesized in shell gland and then deposited onto the eggshell of chickens. Baird et al. (1975) and Yamada (1972) supported the hypothesis that porphyrins in eggshell were most likely to be synthesized in shell gland. Based on the previous reports and our findings, it could be inferred that both biliverdin and protoporphyrin in eggshell were synthesized in shell gland and then were deposited to eggshell simultaneously, and the deposition rate of them both reached the maximal level at the end of eggshell formation.

	ACKNOWLEDGMENTS

 
This work was funded by the Major State Basic Research Development Program of China (2006CB102100).

	FOOTNOTES

 
¹ These authors contributed equally to this work.

Received for publication April 16, 2007. Accepted for publication June 15, 2007.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Baird, T., S. E. Solomon, and D. R. Tedstone. 1975. Localisation and characterisation of eggshell porphyrin in several avian species. Br. Poult. Sci. 16:201–208.[ISI][Medline]

Ito, S., M. Tsudzuki, M. Komort, and M. Mizutani. 1993. Celadon: An eggshell color mutation in Japanese quail. J. Hered. 84:145–147.[Abstract/Free Full Text]

Kennedy, G. Y., and H. G. Vevers. 1976. A survey of avian eggshell pigments. Comp. Biochem. Physiol. B 55:117–123.[Medline]

Poole, H. K. 1965. Egg shell pigmentation of Japanese quail: Genetic control of the white egg trait. J. Hered. 55:136–138.[ISI]

Yamada, M. 1972. d-Aminolevulinic acid dehydratases from shell gland and liver of Japanese quail, Coturnix coturnix japonica. I. Purification, properties and hormonal induction. Biochim. Biophys. Acta 279:535–543.[Medline]

Zhao, R., G. Y. Xu, Z. Z. Liu, X. Y. Li, and N. Yang. 2006. A study on eggshell pigmentation: Biliverdin in blue-shelled chickens. Poult. Sci. 85:546–549.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, X.-T. Articles by Wu, C.-X. Search for Related Content PubMed PubMed Citation Articles by Wang, X.-T. Articles by Wu, C.-X.