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Brazilian Journal of Poultry Science

Print version ISSN 1516-635X

Rev. Bras. Cienc. Avic. vol.13 no.3 Campinas July/Sept. 2011 

Effects of different dietary vitamin combinations on the egg quality and vitamin deposition in the whole egg of laying hens



Zang HI; Zhang KI; Ding XI; Bai SI; Hernández JMII; Yao BIII

IInstitute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Ya'an, Sichuan 625014, P. R. China
IIDSM NUTRITIONAL PRODUCTS Ltd., R&D Animal Nutrition and Health, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland
IIIDSM(China) Limited, No.476, LiBing Road, Zhangjiang High-Tech Park, PuDong Area, ShangHai 201203

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The experiment was conducted to evaluate the effects of different dietary vitamin combinations on the egg quality and vitamin concentrations in the eggs of commercial laying hens. A total of 1,800 25-week-old Lohman pink-shell hens were randomly assigned to four dietary vitamin treatments as follows: NRC(1994) level, NRC (1994) level with Hy.D® (25-hydroxy-cholecalciferol), Local level (current average industry level in China) and OVN® level (optimum vitamin nutrition level), with 10 replicates per treatment and 45 layers per replicate. Hens were housed in commercial laying cages with three birds per cage and given ad libitum access to feed. Results showed the hens that received the fortified vitamin levels in the OVN® treatment had a significantly (p<0.05) lower number of cracked (.47%) and dirty eggs (.27%), and increased egg deposition of vitamin B12, folic acid, vitamin A, vitamin D, 25-OH-D3, vitamin E, vitamin B1, biotin and pantothenate (p<0.05). Treatments had no significant effect on egg-shape index, egg specific gravity, Haugh units and eggshell thickness. Hens fed the NRC-Hy.D® combination also experienced a significant decrease in cracked and dirty eggs (.70% and .44%, respectively) and an increased deposition of 25-OH-D3 in comparison with the NRC treatment. Results of the present study suggest that that the Local treatment was able to improve egg quality parameters of laying hens, but resulted in more cracked and dirty eggs. OVN® reduced the number of cracked eggs and dirty eggs, and improved the deposition of several vitamins in eggs. With the addition of Hy.D®, eggshell strength and 25-OH-D3 deposition in eggs were also improved, and cracked and dirty egg rates declined.

Keywords: Egg quality, Hy.D®, laying hen, OVN®, vitamin deposition.




Vitamins are essential nutrients found in foods. While their requirements are small, they perform specific and vital functions essential for health maintenance. Vitamin deficiencies may lead to a series of diseases in humans. Vitamin A deficiency is the most frequent cause of blindness among pre-school children (Underwood, 1998), abortion of pregnant women and newborn mortality (Radhika et al., 2002) in developing countries. Vitamin D deficiency is a common problem during the winter in Europe, because of the restricted ultraviolet light exposure (Scharla, 1998). A less severe vitamin D deficiency can result in postmenopausal osteoporosis (Lips et al., 2001) and may also increase the risk of initiation and progression of prostate cancer (Tuohimaa et al., 2001). Folate deficiency can result in an increase in plasma homocysteine concentrations, which is linked to an increased risk for cardiovascular disease (Boushey et al., 1995; Refsum et al., 1998), Alzheimer's disease (Morris, 2003), and osteoporosis (Mclean et al., 2004). There is an increased awareness of the importance of this B-vitamin due to the fact that supplemental folic acid has been shown to reduce a woman's risk for having a baby with a neural tube defect (Czeizel & Dudas, 1992; Scott, 1999). Therefore considerable attention has been given to the development of strategies to increase human vitamin intake.

Poultry products contribute significantly to the vitamin intake of consumers. Eggs are one of the most common daily foods, and naturally contain most of the recognized vitamins (vitamin A, vitamin D, vitamin E and B-vitamins), except for vitamin C. Improvements in egg nutritional value may have direct positive implications for daily nutrient intake and consequently for human health (Nys & Sauveur, 2004). Vitamin-enriched eggs are attractive as a vehicle that can provide consumers with compounds that may be beneficial to health or to over come nutritional imbalances. Like many other nutrients, vitamin levels incorporated into feed directly influence the deposition of vitamins in eggs (Naber & Squires, 1993). Vitamin concentration is influenced by genetics, egg production rate and, similarly to fatty acids, it varies with the composition of the hens' diet (Naber, 1993; Leeson & Caston, 2003). With a moderate enrichment of vitamins in accordance with the recommendations for fortification of foods, eggs could play an important role as a functional food. Numerous relevant data related to egg vitamin enrichment through nutrition strategies are now presented, with special attention paid to fat-soluble vitamins E, A, D, but also to vitamin B12 and folic acid. Naber (1993) summarized his findings, classifying the transfer efficiency of vitamins from the diet of hens to eggs, which was very high for vitamin A; high for riboflavin, pantothenic acid, biotin, and vitamin B12; medium for vitamin D3 and E; and low for vitamin K, thiamine and folacin; however, the combined effects of vitamin premix on vitamin deposition in eggs are rarely reported. Therefore, the aim of the present study was to investigate the possibility of enhancing vitamin accumulation in eggs through their supplementation in commercial laying hen diets and to determine the effects of these vitamins on egg quality parameters.



This animal experiment was conducted in accordance with guidelines approved by Animal Health and Care Committee of Sichuan Agricultural University and was performed in accordance with recommendations of the China Council on Animal Care as specified in the Guide to the Care and Use of Experimental Animals.

Animals and Housing

A total of 1,800 25-week-old Lohmanpink-shell commercial laying hens were randomly assigned to four dietary treatments, with 10 replicates per treatment and 45 layers per replicate. All hens were housed in commercial laying cages with three birds per cage (40×35×40cm) according to a randomized complete block design at the Animal Nutrition Research Centre of Sichuan Agricultural University for a 39-wk trial. Hens received 16 h/d of manipulated lighting and ventilation at a natural ambient temperature. Feed and water were provided ad libitum throughout the experiment.

Experimental design and diets

The single factorial design consisted of four vitamin levels (as shown in Table 1): NRC (1994) level, NRC (1994) level with Hy.D® (25-hydroxy-cholecalciferol), Local level (the current average industry level in China) and OVN® level (optimum vitamin nutrition level), the supplemental level of each of them in basal diet was 400 mg/kg. The basal diet was a corn-soybean meal-rapeseed meal layer diet formulated according to the recommendations of NRC (1994) (Table 2). Vitamins were supplied by DSM (DSM China Ltd Corp, Shanghai, P. R. China).





Data collection

The number of dirty and cracked eggs were recorded daily by replicate and determined on the basis of each replicate weekly. Every four weeks of the trial, 10 eggs per replicate were randomly collected for the measurement of egg-shape index (ESI), egg specific gravity (ESG), eggshell strength (ESS), eggshell thickness (EST) and Haugh unit (HU). Egg specific gravity was determined by using the saline flotation method as described by Hempe et al. (1988). Haugh unit was calculated using the formula of Eisen et al. (1962) based on the height of albumen determined by a micrometer and egg weight. The eggshell strength and thickness were determined using the eggshell strength meter and eggshell thickness gauge (Fujihira Corp., Tokyo, Japan) respectively. After the last eggshell quality measurements were taken, the entire contents of eggs were collected and homogenized on the basis of each replicate. Samples were frozen and then freeze-dried to constant weight. Subsamples were reground and then allowed to equilibrate with ambient moisture at room temperature. Samples were sent to Analytical Research Center of DSM Nutritional Products Ltd (Switzerland) to determine the content for all vitamins using HPLC.


Statistical analyses

Data were analyzed by one-way analysis of variance using ANOVA procedure of SPSS (SPSS11.0, 2001) and significant differences among treatment means were compared using the least significant difference test. Statements of statistical significance were based on a probability of (p<0.05).



Egg quality parameters, cracked egg and dirty egg of laying hens

As shown in Table 3, the effects of different dietary vitamin levels exhibited did not result in significant differences in the egg quality parameters measured, such as ESI, ESG, HU and EST (p>0.05); however, ESS in the Local group (4.33 kg/cm2) was significantly higher than the other treatments (4.06, 4.07, 3.92 kg/cm2 for OVN®, NRC with Hy.D®, and NRC level, respectively) (p<0.05). CEP and DEP in the vitamin fortified group (OVN®) was significantly reduced (p<0.05) as compared to other treatments. As for NRC with Hy.D®, with the addition of Hy.D® in the diet, CEP and DEP significantly declined. Furthermore, ESS was significantly improved (p<0.05), as compared with NRC or Local level diet.



Vitamin concentration in whole eggs

Results of vitamin concentration in whole eggs were presented in Table 4. Concentrations of vitamin B12, vitamin A, vitamin E, vitamin B1 and pantothenate in whole egg in the OVN® group were all significantly higher in comparison with Local, NRC with Hy.D® and NRC groups (p<0.05). Folic acid and vitamin D concentrations for OVN® and Local levels were both significantly higher than NRC with Hy.D® and NRC levels (p<0.05), while no significant difference was observed between them (p>0.05). Vitamin B2 concentrations were similar among the four dietary treatments with no significant difference (p>0.05). 25-OH-D3 concentration for the OVN® level was significantly higher than for the NRC level (p<0.05), but no significant difference was observed as compared with Local level or NRC level with Hy.D®. As for NRC level with Hy.D® and NRC level, there was no significant difference for most of the vitamins, except for 25-OH-D3 concentrations, which improved in the NRC level with Hy.D® group. Vitamin B6 and vitamin K concentrations in eggs were not determined because they were below the detection limit.




As compared to the Local level, NRC level with Hy.D® and NRC level, the fortified vitamins in the OVN® diet significantly decreased CEP and DEP. Hens receiving only NRC vitamins had an extremely high CEP and DEP, but this was reduced with the addition of Hy.D®. Furthermore, the ESS was significantly improved, suggesting the inability of regular vitamin D to support maximal shell quality in eggs. The results of the present study were in accordance with the report of Soto-Salanova & Hernandez (2004) and Soto-Salanova & Molinero (2005). It was clear that for NRC level with Hy.D® and NRC level, the only difference between them was Hy.D® inclusion, and no other dietary difference. The OVN® diet still contained vitamin C except for Hy.D®. Hy.D® is a metabolite of vitamin D3, 25-OH-D3 ÿwhich is the most abundant circulating form of vitamin D and plasma levels of the metabolite gives a good indication of the vitamin D status of the chick (Haussler & Rasmussen, 1972). Positive effects of 25-OH-D3 on egg production and quality have been reported and summarized by Soares et al., (1995). Fritts & Waldroup (2003) showed that at comparable levels of potency, 25-OH-D3 was more efficacious than vitamin D3 in terms of promoting bone and shell formation. Keshavarz (1996) reported that diets containing 6.25 µg vitamin D3/kg feed increased the incidence of cracked eggs and eggshell deformations. Poor shell quality is normally a result of poor calcium, phosphorus and vitamin D metabolism. Birds receiving Hy.D® were able to maintain their plasma 25-OH-D3 at a higher level, thus helping to maintain their bone integrity, egg production and eggshell quality.

In addition to the observed changes in eggshell strength, vitamin C may play some role in improving bone properties and eggshell formation (Sergeev et al., 1990; Orban et al., 1993). Vitamin C is required for the conversion of vitamin D into its metabolite form calcitrol, which is essential for calcium regulation and the calcification process (Thornton, 1970). Vitamin C is an essential cofactor in the formation of collagen and of the extracellular matrix (Newman & Leeson, 1997). This improvement may be attributed to increased calcium absorption, or possibly to the role that vitamin C plays in the development of bone tissue.

Most reports about the effect of vitamin on egg quality focus on fat-soluble vitamins, while a few reports are on water-soluble vitamins, which also present different results. Keshavarz (2003) reported that supplementing vitamin D or 25-OH-D3 in the diet did not influence specific gravity of eggs from hens from 49 to 65 wk of age. Mattila et al. (2004) reported that no major differences in Haugh units, eggshell strength or specific gravity were observed between the control and vitamin D2- or D3-enriched diets. On the other hand, Atencio et al. (2006) reported that eggs from hens fed the lowest vitamin D3 levels had lower specific gravity than those from hens fed the highest vitamin D3 levels. Mendonca et al. (2002) and Mori et al. (2003) reported that the eggshell index, specific gravity, shell thickness and albumen quality of eggs obtained from hens fed supplemental vitamin A or vitamin A and E did not differ from those laid by hens fed the basal diet. Puth pongsiriporn et al. (2001) showed that dietary vitamin E and C supplementation had no significant effect on eggshell or Haugh units. Radwanet al. (2008) reported that the addition of vitamin E (100 or 200 mg/kg diet) in laying hens diets did not significantly affect eggshell thickness, egg shape index or Haugh units. Sahinet al. (2002), however, reported that egg specific gravity, eggshell thickness and Haugh unit were positively influenced by vitamin E supplementation in Japanese quails. Alteration of dietary vitamin level had minor effects on egg quality parameters in terms of the egg shape index, specific gravity, shell thickness and Haugh unit in the present study, which was in agreement with most of reports discussed above.

Vitamin concentration in the hen diet is the most important factor in determining vitamin content in the egg. As vitamin levels in the diet are increased, there is an increase in vitamin levels deposited in the albumen and/or yolk. Supplementation with increasing amounts of dietary vitamins for OVN® level produced significant improvement in egg vitamin concentrations in this present study, which is consistent with the report of Perez-Vendrellet al. (2003), who conducted a study in layers using fortified vitamin levels or those commonly used in Spain. For most of the vitamins there were increased accumulations in the egg in response to higher dietary supplementation. Some promising results have been highlighted by Leeson & Caston (2003) for the potential transfer efficiency of some vitamins from hen diets to the egg. These authors studied the effect of supplementing the diet with two vitamin premixes (the regular level and enriched level), and results showed that the concentration of vitamin B12 in enriched eggs significantly increased from 36 to over 100% DRI in response to an 11-fold increase in diet vitamin supplementation. There was also meaningful egg enrichment for vitamin D3 and E, and the level of pantothenic acid was doubled, while vitamin K and biotin levels in modified eggs were not significantly different to those observed in regular eggs.

In the present study, Local level and OVN® level significantly improved the concentrations of vitamin A, vitamin D3, vitamin E, folic acid and pantothenic acid in eggs as compared to NRC level with Hy.D® and NRC level. Also the OVN® level produced significantly higher levels of vitamin A, E and pantothenic acid than the Local level. These results were in agreement with other investigators who reported the improvement of certain vitamin deposition in eggs when the hen diet was supplemented with a high dose of vitamin A (Squires & Naber, 1993; Jiang et al., 1994; Surai et al., 1998), vitamin D3 ( Mattila et al., 1999; Mattila et al., 2003; Mattila et al., 2004), vitamin E (Frigg et al., 1992; Jiang et al., 1994; Qi & Sim, 1998; Galobart et al., 2002; Grobas et al., 2002), folic acid (House et al., 2002; Hebert et al., 2005; Tactacan et al., 2010) or pantothenic acid (Leeson & Caston, 2003). Mendonca et al., (2002) reported that progressive increases in the incorporation of retinol into egg yolk was verified when vitamin A was supplemented to the basal diet; egg yolk retinol content increased linearly as dietary vitamin A increased. Mori et al. (2003) reported yolk retinol concentration was enhanced by added vitamin A, from 24.6 IU/g for eggs from the control group, to 33.6 and 37.7 IU/g of yolk when hens were fed 15,000 and 30,000 IU/kg of diet. Yolk αα-tocopherol was significantly increased by dietary tocopherol supplementation, ranging from 10.9 µg/g (control group) to 160.6, 264.1, and 383.2 µg/g of yolk, respectively, when 200, 400 and 600 mg/kg of ration were added, respectively. Jiang et al. (1994) reported that egg yolk αα-tocopherol level linearly increased as dietary tocopherol increased. Pal et al. (2002) also showed that using 110 vs. 55 IU vitamin E /kg diet doubled egg vitamin E content. Dickson et al. (2010) concluded that eggs of laying hens were consistently enriched with folate by the dietary supplementation with 4 mg of folic acid/kg of diet throughout the production cycle. Hoeyet al. (2009) and Bunchasak & Kachana (2009) reported that it was possible to use synthetic folate at high doses (16 and 10 mg/kg diet) to produce novel eggs enriched with natural folates.

There was a significant increase in egg 25-OH-D3 concentration with the addition of Hy.D® to NRC level diet. This result was not consistent with Mattila et al. (1999), who reported a strong positive correlation between cholecalciferol content in poultry feed and cholecalciferol (r = 0.995) and 25-hydroxycholecalciferol (r = 0.941) contents in egg yolk. Ovesen et al. (2003) also suggested that vitamin D in eggs is present almost exclusively as 25-hydroxycholecalciferol, which is absorbed better and faster and has greater biological activity than cholecalciferol. Meanwhile, there was also a significant increase of in egg enrichment of vitamin B1, vitamin B12 and biotin in OVN® level, whereas the Local level did not produce any changes. Vitamin B12 perhaps showed the best response, and this finding was in agreement with Squires & Naber (1992), who reported that egg yolk vitamin B12 concentration rapidly responded to dietary changes in the levels of this vitamin and was indicative of the vitamin B12 status of the hen. Concentration of biotin in egg albumen increased with incremental dietary biotin levels, but egg yolk concentration was stable, and positive relationship between dietary biotin and the amount of biotin in eggs was also observed (Robel, 1991). The concentration of riboflavin in the eggs of hens fed the OVN® level and the Local level was higher than with the NRC level with Hy.D® and NRC level, but no significant effect was observed. Naber & Squires (1993) reported that there was nearly a linear relationship of diet riboflavin to egg riboflavin contents in the range of 1.5 to 5.0 mg/kg of feed. At 2 to 4 times the dietary requirement of the hens, riboflavin deposition in the eggs was limited by the transfer of riboflavin into the ovum (Squires & Naber, 1993). Riboflavin deposition in the eggs was dependent on dietary riboflavin and reached half-maximal values at about 2 mg of supplemental riboflavin (White et al., 1986).

Concentrations of vitamin B6 and vitamin K3 were not obtained because they were below the detection limit level in the present study. Considering its restricted biological role, very few studies have attempted to enhance its concentration in eggs. Suzuki & Masayuki (1997) studying the increasing number of hemorrhagic diseases of newborn babies in relation to vitamin K deficiency of pregnant women, demonstrated that by feeding hens with high doses of either phylloquinone (vitamin K1), menaquinone (vitamin K2) or menadione (vitamin K3), it was possible to increase the level of both K1 and K2 up to 1,908 and 240 µg/100 g egg yolk, respectively.



In conclusion, results of the present study demonstrated that hens receiving vitamins only at NRC levels presented an extremely high cracked egg and dirty egg rates, but this was reduced with the addition of Hy.D®. The addition of Hy.D® significantly enhanced eggshell strength and 25-OH-D3 concentration in the egg. Hens receiving the Local vitamin levels produced more cracked eggs, dirty eggs and lower vitamin levels in eggs. Hens receiving OVN® vitamins produced eggs containing higher levels of most vitamins, with the least impact seen for dirty and cracked eggs. Apparently, proper vitamin nutrition has significant implications in gastrointestinal health of hens. This could have positive ramifications in marketing eggs with a higher vitamin level and a better nutritive value. The outcomes of this study will enable layer farmers to use vitamins more effectively for the development of functional eggs to meet the needs of specific individuals.



The authors would like to acknowledge the financial support of DSM (China) Ltd and DSM Nutritional Products Ltd (Switzerland).



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Mail Adress:
K Zhang
Institute of Animal Nutrition
Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education
Sichuan Agricultural University
Ya'an, Sichuan 625014, P. R. China
Tel: +86-13981605686
Fax: +86-835-2885630

Submitted: November/2010
Approved: June/2011

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