INTRODUCTION
Vitamins are organic compounds required in minute amounts for normal metabolism and production in animals [1]. Because vitamins cannot be synthesized by host animals to meet their requirements, they must be supplied through diets [2]. Most feedstuffs used for poultry diet are rich in energy and protein but lack sufficient amounts of micro element or certain vitamins [3]. In addition, the availability of different vitamins in feedstuffs varies considerably [4]. Vitamin analysis in feedstuffs is time consuming and costly. Thus, vitamin premix is usually supplemented at recommended levels. Normally vitamin premix contains 12 essential vitamins for poultry to meet recommended levels of vitamins and ensure normal function and productivity [5].
Vitamin premix needs to be applied in adequate amounts without deficiency or adverse effects on productivity [6]. Recently, the supply and price of vitamins are changing rapidly in the global market. Thus, attention is focused on applying appropriate levels of vitamin premix into poultry diets because vitamins are among the most expensive feed ingredients. Some studies have determined effects of vitamin premix removal on growth performance of broiler chicks. Chickens fed diets lacking vitamin premix exhibit reduced weight gain, feed efficiency, and survival rate [7]. On the contrary, Moravej et al. [8] have reported that reduction or withdrawal of vitamin premix in broiler diets does not affect growth performance during the finishing period. Such discrepancy might be due to differences in raw material compositions of basal diet and duration of withdrawal. Furthermore, only limited information is available on dietary effects of reduction or withdrawal of vitamin premix on egg production, egg qualities, and nutritional quality of eggs in commercial layers. Thus, this study was conducted to determine effects of absence or reduction of vitamin premix in corn soybean meal-based layer diet on egg production, egg quality, and some vitamin contents in chicken eggs.
MATERIALS AND METHODS
A total of 144 45-week-old Hy-line brown layers were randomly assigned to four groups (36 layers for each group) so that egg production was similar for each group. They were fed diets with different levels of vitamin premix during eight weeks. Treatments consisted of four experimental diets containing 1,000 mg, 750 mg, 500 mg, or 0 mg vitamin premix/kg of diet. Laying hens in the control group received diet with 100% vitamin premix (0.1% in experimental diet). The other two groups received a diet containing 75% or 50% of vitamin premix compared to control, respectively. The fourth group of layers received the basal diet without supplemental vitamin premix. Layers were housed in wire cages. Each replicate comprised six cages with six birds in each cage. The formula and chemical compositions of experimental diets are shown in Table 1. All diets were formulated to meet and exceed the nutrients requirements of NRC [3]. Experimental diets and water were provided for ad libitum consumption. Vitamin contents in premix are described in Table 2. A room temperature of 22 ± 3°C and an artificial lighting of 16 h were maintained throughout the entire experimental period. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Konkuk University (KU21140).
Control, a group received a diet containing 0.10% vitamin premix; T1, a group received a diet containing 0.075% vitamin premix; T2, a group received a diet containing 0.05% vitamin premix; T3, a group received a diet without any vitamin premix.
Vitamin mixture provided the following nutrients per kg of control diet: vitamin A, 10,000 IU; vitamin D3, 3,500 IU; vitamin E, 10 mg; vitamin K3, 3 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; vitamin B6, 4 mg; vitamin B12, 0.0235 mg; pantothenic acid, 10 mg; folic acid, 0.82 mg; nicotinic acid, 30 mg.
Diets were freshly added every day and feed intake was recorded weekly by replicate. Eggs were collected at a fixed time every day. The number and weight of eggs laid were recorded every day during the experimental period. Abnormal eggs were excluded from egg weight measurement. Egg mass was calculated as hen-day egg production multiplied by the average egg weight.
Egg quality was determined bi-weekly during the post-molting experiment (at 2, 4, and 6 week of experiment). Five eggs from each replicate were collected, weighed, and stored overnight at room temperature (20°C) for subsequent analyses. The breaking strength of each sampled egg was measured using a DET-6000 digital egg tester (Nabel, Kyoto, Japan). Eggshell thickness without shell membrane was measured using a micrometer (series 547-360, Digimatic micrometer, Mitutoyo, Japan). Egg yolk color was determined by comparison with a Roche yolk color fan (Hoffman-La Roche, Basel, Switzerland). Albumen height was also measured using the DET-6000 digital egg tester (Nabel). Haugh unit calculation was then performed.
At the end of the experiment, one bird was randomly selected for each replicate and blood was drawn from a wing vein. Serum was then obtained after a gentle centrifugation (2,000×g for 15 min). Serum samples were stored at −20°C until analysis. Levels of serum albumin, globulin, total cholesterol, and triacylglycerol were measured using an automatic blood analyzer (Labospect 008AS, Hitachi, Tokyo, Japan). Glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) levels in serum samples were measured according to a colorimetric method as previously described [9].
All eggs laid on the last day of the experiment were collected. Two eggs obtained from each replicate were broken. Egg yolk was separated from the respective egg white and homogenized using a food blender for α-tocopherol analysis. Another two eggs also were broken and whole eggs were used for riboflavin analysis. An HPLC (Nexera 40 series UHPLC, Shimadzu, Tokyo, Japan) equipped with a diode array detector (DAD), a autosampler, a dual pump, and a YMC C30 column (150 × 4.6 mm, 3 µm; YMC, Wilmington, NC, USA) was used for α-tocopherol analysis as previously described [10]. Samples were scanned (180–800 nm) with a 0.05 min (1 s) response time at a detection wavelength of 295 nm. An HPLC (Nanospace SI-2, OSAKA SODA, Tokyo, Japan) equipped with an autosampler, a pump, a UV detector, and a PDA detector system was used for riboflavin analysis.
Data were analyzed by the GLM procedure of SAS with cage lot (six adjacent cages) as experimental unit for evaluating egg production, egg quality, and vitamin contents in eggs. Individual layers were considered as unit for blood profiles. Orthogonal polynomial contrasts were used to determine linear and quadratic effects of dietary vitamin premix levels on responses measured. Statistical significance was accepted at p < 0.05.
RESULTS AND DISCUSSION
Laying performances of hens fed diets with varying levels of vitamin premix are presented in Table 3. There was no significant difference in egg production or daily egg mass among groups during the first four weeks of experiment. Egg production and daily egg mass in the control group were significantly higher (p < 0.01) than those in hens fed diet without vitamin premix during the second half of the experiment (the second four weeks). A linear trend for egg production with increasing dietary vitamin premix levels was found during the same period. Egg weight and feed intake were not affected by dietary treatment.
As shown in Table 4, diets with varying levels of vitamin premix did not influence any egg quality parameters when measured at 2 weeks of the experiment. A linear trend for yolk color with increasing dietary vitamin premix level was found during the same period. Eggshell strength and thickness for hens fed diet without vitamin premix were significantly lower (p < 0.05) than those for hens in the other three groups at 4 and 6 weeks of the experiment. Significant linear and quadratic improvement for eggshell strength and thickness with increasing dietary vitamin premix levels (p < 0.05) were found. However, Haugh unit was not affected by dietary treatment.
Results from broilers regarding effects of vitamin premix withdrawal on growth performance are conflicting. Chickens fed diets lacking vitamin premix exhibit reduced weight gain, feed efficiency, and survival rate in some studies [7]. Conversely, Moravej et al. [8] did not find any significant effects of reduction or withdrawal of vitamin premix on growth performance during the finishing period. Information is limited concerning effects of reduction or withdrawal of vitamin premix on egg production and egg quality measurements in commercial layers. It appears that withdrawn of vitamin premix, especially riboflavin, negatively affects laying performance [11]. Long-term riboflavin deficiency can reduce egg production, egg weight, and body weight of layers [12]. Our results revealed that reduction more than 50% or withdrawal of vitamin premix in layer diet did negatively affect egg production and egg qualities as the period of deprivation increased. Most vitamins are not stable. They can undergo significant deterioration during storage [5]. Vitamin availability in plant feedstuffs is often very low [13]. The calculated riboflavin value of basal diet used in this study appears to barely meet the riboflavin requirement [3,5]. Considering these, a part of the decline in laying performance might be due to insufficient supply of riboflavin.
Blood profiles of hens fed diets with varying levels of vitamin premix are presented in Table 5. There were no significant differences in concentrations of albumin, globulin, total cholesterol, or triacylglycerol among groups. A linear trend for serum albumin with increasing dietary vitamin premix level was found. Activities of serum GOT and GPT as indicatives of tissue damages were not affected by dietary treatment.
In most studies, blood biochemistry and immune criteria of poultry were not affected when vitamin premix was reduced or removed from experimental diets. For example, Deyhim and Teeter [7] did not find any significant effects of reducing vitamin premix on humoral immune response. In addition, removal of vitamin premix in broiler diets had no adverse effect on immunocompetence or antibody titer production over a relatively short period [14]. In the present study, relative weights of thymus and bursa of Fabricius were not influenced by dietary treatment (data not shown). Studies about long-term effects of vitamin premix withdrawal on immune criteria and blood biochemistry in commercial laying hens are limited. The possibility of impaired immune index should not be precluded. Further studies are needed to clarify effects of diets containing various levels of vitamin premix on immune criteria of commercial haying hens.
Contents of riboflavin and α-tocopherol obtained from hens fed diets containing varying levels of vitamin premix are presented in Table 6. The level of α-tocopherol of eggs obtained from the control group was significantly higher than those of groups with vitamin premix reduction of more than 50% or withdrawal of vitamin premix (p < 0.001). A significant linear increment for egg α-tocopherol with increasing dietary vitamin premix level was found. The level of riboflavin of eggs obtained from control group was significantly higher than that of the group fed diet without vitamin premix (p < 0.05). Similarly, a linear trend for riboflavin content with increasing dietary vitamin premix level was found.
Naber and Squires [11] have reported that riboflavin levels are dropped within several days in egg from layers fed diets without riboflavin or all supplemental vitamins. Another study has also found that egg riboflavin concentration is rapidly decreased after feeding diets without riboflavin supplement [12]. These studies have suggested that riboflavin concentration in egg albumen can be used to assess nutritional status of laying hens [11,12]. In the present study, the riboflavin content of eggs was affected by dietary levels of vitamin premix, in agreement with those results. As expected, levels of α-tocopherol in experimental diets were reflected in concentrations of this vitamin in egg obtained each group. Scheideler et al. [15] have reported that egg yolk α-tocopherol is increased linearly with vitamin E supplementation, although dietary levels are not different from those in the present study. Chicken eggs are one of the most common daily foods. They contain most of the essential vitamins except for vitamin C [16]. Vitamin E is a natural fat-soluble antioxidant that tends to bring about storage stability of eggs [17]. Eggs low in riboflavin and α-tocopherol are less attractive to consumers who consider their health.
CONCLUSION
Overall, reducing more than 50% or withdrawal of vitamin premix in layer diet did negatively affect egg production or egg qualities. From results of the present study, it is recommended to feed a layer diet containing sufficient levels of vitamin premix to produce healthy chicken eggs with a long shelf life.
