Effect of Vitamin D2- and D3-Enriched Diets on Egg Vitamin D Content, Production, and Bird Condition During an Entire Production Period P. Mattila,*,1 J. Valaja,† L. Rossow,‡ E. Vena¨la¨inen,† and T. Tupasela* *MTT Agrifood Research Finland, Food Research, FIN-31600 Jokioinen, Finland; †MTT Agrifood Research Finland, Animal Nutrition, FIN-31600 Jokioinen, Finland; and ‡National Veterinary and Food Research Institute (EELA), FIN-00581 Helsinki, 15 Finland ABSTRACT Vitamin D insufficiency during winter is a common problem for humans in Europe. One way to ease this problem is through the production of vitamin Dfortified eggs. To evaluate such a production process, the effects of vitamin D supplementation during an entire production period were assessed. Transfer of vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) from the diet to egg yolks was measured using 2 different levels of both vitamins (6,000 and 15,000 IU/kg feed) relative to a control treatment (2,500 IU vitamin D3/kg feed). During the experiment, production parameters, egg quality (egg weight, Haugh unit, specific gravity, eggshell fracture force, and Ca content of eggshell), and the condition of hens were monitored. At the end of the experiment histopathological tests were performed. Supplementing
diets with vitamin D3 increased egg yolk vitamin D content more effectively than did supplementation with vitamin D2. For groups of hens receiving 6,000 or 15,000 IU of vitamin D3/kg feed, egg yolk vitamin D3 content ranged from 9.1 to 13.6 and from 25.3 to 33.7 µg/100 g, respectively. Corresponding values for birds fed vitamin D2 were 4.7 to 7.0 and 13.3 to 21.0 µg/100 g. Both supplements enhanced vitamin D3 content of egg yolks relative to the control diet (2.5 to 5.0 µg/100 g of egg yolk). Vitamin D supplements had no effects on production parameters compared with the control diet. However, especially vitamin D3 improved bone strength (P < 0.05). Autopsy at the end of the experiment indicated no detrimental accumulation of calcium in the kidneys, liver, heart, muscles, or lungs.
(Key words: egg quality, egg yolk, hen, vitamin D2, vitamin D3) 2004 Poultry Science 83:433–440
D insufficiency in the winter (Scharla, 1998; Romagnoli et al., 1999; Brot et al., 2001; Lamberg-Allardt et al., 2001). Eggs are among the few potent natural sources of vitamin D for humans. Recent research has indicated that the vitamin D3 (cholecalciferol) content of eggs can be further increased by supplementing hen feed with vitamin D3. Provision of additional vitamin D3 diets does not alter the fatty acid composition or the sensory and functional properties of eggs. Furthermore, inclusion of vitamin D3 up to 12,000 IU/kg feed used in these relatively shortterm studies did not appear to be harmful to hens (Mattila et al., 1999, 2003). The present study is a continuation of the previous surveys (Mattila et al., 1999, 2003) examining the possibility of fortifying eggs with vitamin D. The special aim of the present study was to assess the effects of vitamin D2 and D3 supplementation during an entire egg-laying period on production, egg quality, and hen health and condition. Observations of hen health were confirmed by histopathological tests at the end of the study.
INTRODUCTION Vitamin D is crucial for normal bone formation. A deficiency, when prolonged and severe, can lead to osteomalacia in adults or rickets in children. Less severe vitamin D deficiency (vitamin D insufficiency) can result in postmenopausal osteoporosis (Eastell and Riggs, 1999; Lips et al., 2001) and may also increase the risk of initiation and progression of prostate cancer (Tuohimaa et al., 2001). Vitamin D can be derived from the diet or produced in the skin by the action of sunlight. The intake of vitamin D is low in most countries because few foods are relatively rich in vitamin D. Vitamin D insufficiency is a common problem during the winter in Europe, because the continent is located at a high latitude leading to restricted ultraviolet light exposure (Scharla, 1998). As a result, population groups including adolescents, young adults, middle-aged women, and elderly people are at risk of vitamin
MATERIALS AND METHODS Birds and Housing
2004 Poultry Science Association, Inc. Received for publication July 29, 2003. Accepted for publication October 13, 2003. 1 To whom correspondence should be addressed:
[email protected].
A total of 450, 20-wk-old laying hens (Lohmann LSL White) were divided into 5 feeding groups of 90 individu433
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als and used in a 48-wk experiment. Hens were housed in a windowless room in 2-tier batteries of cages suitable for holding 3 birds each (640 cm2 per hen). Each treatment was offered to 6 cages and was replicated 5 times. Temperature was maintained at 18 ± 2°C. Lighting was controlled using a program of 12.5 h at the start of the experiment and advanced by 30 min/wk until a total of 14.5 h was achieved. Water was provided ad libitum from nipple drinkers.
Experimental Diets and Feeding Prior to the experiment all the hens were offered a conventional diet that contained a standard amount of vitamin D3 (2,500 IU/kg). At the start of the study 5 different treatments were assessed. Treatments consisted of diets containing 2,500 IU of vitamin D3/kg of feed (control), 6,000 IU of vitamin D3/kg of feed (treatment 2), 15,000 IU of vitamin D3/kg of feed (treatment 3), 6,000 IU of vitamin D2 and 2,500 IU of vitamin D3/kg of feed (treatment 4), and 15,000 IU of vitamin D2 and 2,500 IU of vitamin D3/kg of feed (treatment 5). Vitamin D3 was included in treatments 4 and 5 due to the low bioavailability of vitamin D2 (Ameenuddin et al., 1985). Ingredient composition of the experimental diets is shown in Table 1. The experiment was divided into 3 16wk phases. Dietary ME content was 10.7, 10.5 and 10.4 MJ/kg during feeding phases 1, 2, and 3, respectively, and the ratio of CP and ME (14:5) was maintained for the entire study. Diets were formulated to meet nutrient requirements of the NRC (1994) and were offered ad libitum as 4-mm pellets. A vitamin premix provided vitamins in excess of requirements for laying hens (NRC, 1994) and supplied 2,500 IU of vitamin D3, 11,300 IU of vitamin A, and 37 mg of vitamin E. Vitamins D32 and D23 were mixed with a small amount of limestone before incorporation to ensure even distribution in each diet.
Experimental Procedures The 48-wk experiment consisted of 12 4-wk periods. Feed intake was measured on a replicate basis by weighing an amount of feed consumed during each 4wk period. Eggs were collected and recorded daily for each replicate. Half of the birds were weighed at the beginning of each feeding phase (20, 35, and 51 wk of age) and toward the end of the experiment (65 wk of age). Ten eggs per replicate were collected on 3 occasions during the experiment, at 35, 51, and 67 wk of age, for determination of egg quality (shell strength, specific gravity, albumen height, and eggshell calcium content). Eggshell strength was measured as compressive fracture force using a Canadian eggshell Tester4 (Hamilton, 1982). Spe-
2
Lutavit D3 500 S, BASF, Germany. Sigma Chemical Co., St. Louis, MO. 4 OTAL Precion Company Limited, Ontario, Canada. 5 Model 1000R, Lloyd Instruments, Ltd., Fareham, Hampshire, UK. 6 Leco Corporation, St. Joseph, MI. 3
cific gravity was assessed by the Archimedes method (Hamilton, 1982). Albumen height was measured in Haugh units. Three eggshells per replicate were used for calcium content determinations. Eggshells were oven dried overnight at 105°C and were passed through a hammer mill fitted with a 1-mm mesh before analysis. At the end of the trial, 20 birds from each diet were killed by stunning with carbon dioxide and subsequent neck dislocation. Ten birds per diet were assigned to histopathological examinations, and another 10 birds per diet were used for determination of tibia breaking strength. Breaking strength measurements were performed using a modification of the procedures described by Crenshaw et al. (1981). Tibia samples were restrained at each end by supports 4.5 cm apart, and a force was applied at the midspan. Breaking strength was determined using a Lloyd testing instrument5 fitted with a 500-N load cell. We recorded the amount of force applied at a constant speed (50 mm/ min) necessary to break the bone and the run length (mm) before bone breakage. Tibia length and thickness were measured.
Chemical Analyzes Feed samples were collected after experimental diets were mixed. All samples were passed through a hammer mill fitted with a 1-mm mesh before analysis. Crude fat and ash were determined by standard methods (AOAC, 1990), and crude fiber was measured according to the method of Hirsja¨rvi and Andersen (1954). Feed nitrogen content was assessed using a Leco FP 428 nitrogen analyzer.6 Ca concentration of feeds and eggshells and P concentrations of feeds were determined by ICP emission spectrophotometry (Luh Huang and Schulte, 1985). For vitamin D determinations, 20 randomly sampled eggs were collected from each treatment group after 4, 16, 28, and 44 wk into the experiment. Additional samples were also collected for treatment 5 after 3, 6, 9, and 12 d from the start of the experiment. Once collected, egg yolks were separated and pooled according to collection day. Yolks were mixed, packed in plastic bags, and stored at −18°C to await vitamin D analysis. Samples from feed batches were collected at random and submitted for vitamin D determinations to ensure correct formulation of experimental diets. Vitamin D3 and D2 contents were determined in egg yolk, using previously validated methods (Mattila et al., 1992, 1999, 2003) that involved saponification, extraction, purification by solid-phase extraction, semipreparative normal-phase HPLC, and quantification with reversephase HPLC. Vitamin D2 served as an internal standard for vitamin D3 analysis and vice versa. The concentration of internal standard used was dependent on the amount of the vitamin D compound expected in a given sample. Feed vitamin D determinations were performed according to a method validated by Mattila et al. (1992), with the exception of a purification procedure. Following saponification, extraction, and evaporation, the residue was dissolved in 2 mL of n-hexane and purified by semi-
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EFFECT OF VITAMIN D-ENRICHED FEED ON EGG PRODUCTION TABLE 1. Ingredients (%) and chemical composition of experimental diets Feeding phase and age of hens Item
I, 20 to 35 weeks
II, 36 to 51 weeks
III, 52 to 67 weeks
Ingredients1 Barley Wheat Oats Soybean meal Rapeseed oil Monocalcium phosphate Limestone Salt Trace mineral mixture2 Vitamin mixture3 Methionine
30.14 19.70 20.00 17.80 2.50 0.95 8.10 0.38 0.20 0.16 0.07
31.24 19.60 20.00 17.20 2.10 0.95 8.10 0.38 0.20 0.16 0.07
33.40 19.40 20.00 16.00 1.30 0.97 8.10 0.38 0.20 0.16 0.07
Calculated values1 AME, MJ/kg Crude protein Calcium Available phosphorus
10.7 15.5 3.34 0.38
10.6 15.4 3.34 0.38
10.4 15.1 3.34 0.38
Measured values4 Ash Crude protein Crude fat Crude fiber Calcium Total phosphorus
9.62 17.79 6.01 5.08 3.87 6.3
12.10 18.27 4.73 4.46 3.69 6.0
1
As-fed basis. Trace mineral premix supplied the following per kilogram of diet: 0.63 g of calcium, 29.1 mg of iron, 8.0 mg of copper, 50.3 mg of manganese, 65.1 mg of zinc, 0.51 mg of iodine, and 0.20 mg of selenium. 3 Vitamin premix supplied the following per kilogram of diet: 443.2 mg of calcium, 3.4 mg of retinol, 0.062 mg of cholecalciferol, 37.2 mg of α-tocopherol, 4.9 mg of phylloquinone, 2.2 mg of thiamin, 4.9 mg of riboflavin, 3.4 mg of pyridoxine, 0.016 mg of cyanocobalamin, 0.248 mg of biotin, 0.584 mg of folic acid, 39.3 mg of niacin, 9.8 mg of pantothenic acid, 2.7 mg of canthaxanthin, 6.9 mg of xanthophylls, and 0.49 mg of antioxidants. 4 DM basis. Mean of 5 measurements per feeding phase. 2
preparative HPLC. The solid-phase extraction purification procedure was therefore omitted in the case of feeds. Measurements were performed on feed and egg yolk samples of 2 to 10 g and 10 g, respectively. Semipreparative purification of extracts was carried out using a chromatograph7 fitted with a µ-Porasil column.8 In the analytical step, a Vydac 201 TP54 column9 was used. Measurements were performed in duplicate. In addition, samples were also analyzed in the absence of an internal standard to ensure that other vitamin D compounds or interfering compounds did not coelute with the internal standard peak.
Histopathological Tests Pathological tests were performed on all hens that died during the experiment and for 10 randomly selected birds from each treatment at the end of the feeding study. Nutritional status, skeletal condition, and calcification of soft tissues (kidney, liver, heart, muscle, and lung) were evaluated macroscopically. In addition, liver and kidney condition was also assessed histologically.
7
200 Series, Perkin Elmer, Boston, MA. 10 µm, 300 × 3.9 mm; Millipore Corp., Milford, MA. 5 µm, 250 × 4.6 mm; The Separation Group, Hesperia, CA.
8 9
Statistical Analysis Data were subjected to analysis of variance using the GLM procedure of SAS (SAS Institute, 1990). Production parameters (egg laying and feed intake) were analyzed according to analysis of variance for repeated measurements using the following model: Yijk = µ + di + eij + pk + (p × d)ik + eijk, where Yijk = observation, µ = general mean, di = effect of diet (i = 1,…,5), pk = effect of period (k = 1,…,12), eij = the error term of diet, and eijk = the error term of repeated effect. Egg quality, tibia measurements, weight gain, and mortality were analyzed according to one-way analysis of variance using the following model: Yijk = µ + di + eij, where Yijk = observation, µ = general mean, di = effect of diet (i = 1,…,5), and eij = the error term of diet. In all analyses, one experimental unit consisted of 6 adjacent cages of 3 birds. Diet effects were further separated into 4 single degree of freedom orthogonal contrasts as follows: C1: control treatment vs. vitamin D supplemented diets (group 1 vs. groups 2, 3, 4, and 5), C2: comparison vitamin D3 vs. vitamin D2 supplements (groups 2 and 3 vs. 4 and 5), C3: effect of level of vitamin D supplementation (groups 2 and 4 vs. 3 and 5), and C4: interactions between contrasts C2 and C3 (groups 2 and 5 vs. 3 and 4). In all cases, residuals were plotted against fitted values to ensure normality of experimental data.
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MATTILA ET AL. TABLE 2. Addition and concentration of vitamin D2 and D3 in experimental diets (IU/kg) Feeding phase and treatment
Added vitamin D2
Measured vitamin D2
15,000
13,100
Feeding phase I Treatment 2 Treatment 5 Feeding phase II Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5
6,000 15,000
Feeding phase III Treatment 1 Treatment 3 Treatment 4 Treatment 5
Added vitamin D3
Measured vitamin D3
6,000
5,100
2,500 6,000 15,000
2,600 5,800 13,200
2,500 15,000
3,000 13,900
5,200 13,800
6,000 15,000
5,000 12,600
RESULTS AND DISCUSSION For each of the 3 feeding phases, diets were manufactured as 3 separate batches, and hens were fed each batch for 16 wk. In addition to a basal diet (typical vitamin D content 2,500 IU/kg), 2 different levels of vitamin D2 and vitamin D3 supplements were included (6,000 and 15,000 IU/kg). Minor differences (
