By: Dr. majeda alghunmeen – Jordan

Introduction :
There are two different categories for enhancing vitamin D level in eggs:

enriched eggs: the laying hens that produced these eggs were exposed to sunlight or were fed in a natural way that leads to increased levels of vitamin D in the eggs naturally.
fortified eggs: that vitamin D has been artificially added to laying hens feed during its production period.
in this report we focused on fortified eggs (artificially added vitamin D)
Feeding hens with higher levels of vitamin D3 and 25(OH)D3 has been shown to significantly increase the vitamin D content in egg yolks. Studies have demonstrated that eggs can be fortified to contain between 100 and 500 IU of vitamin D, meeting the recommended daily intake for both children and adults without affecting egg production or quality
These method has proven successful in significantly increasing the vitamin D content of eggs . Consuming these enhanced eggs can help maintain adequate vitamin D levels in humans, providing a valuable dietary strategy to combat vitamin D deficiency.
(Citations·R. Adhikari et al. 2019, 2004 , Citations·Jing Guo et al. 2017 )

Key Notes:
• Fortification Strategy: The hens' diet must include consistent levels of vitamin D3 or 25-hydroxyvitamin D3 (a more bioavailable form). Variations in feed quality can impact the results.

How to assist the level of vitamin D in fortified eggs :
To assess the level of vitamin D in fortified eggs, you should monitor hens at peak production age, as this is when their egg-laying is most consistent, and the vitamin D content is likely stabilized.
Continuous monitoring of the level of vitamin D along all stages in the life cycle hens.

Recommended Age for Checking Vitamin D Levels in Fortified Eggs
1. Peak Production (24-30 weeks of age):
o Hens typically start laying eggs around 18-20 weeks, and egg production peaks around 24-30 weeks of age. At this stage, the hens’ metabolism is most active in terms of egg production and nutrient transfer to eggs. It is the optimal time to assess vitamin D levels in eggs, as fortification should be most effective during peak production.
o Research shows that egg vitamin D content is most stable and responsive to dietary adjustments during this period (Khachik et al., 2004; Bie et al., 2019).

2. Mid-Laying Period (40-50 weeks of age):
o By this age, hens’ egg production may begin to decline slightly, but they are still producing at high levels. Monitoring at this stage helps to track the consistency of vitamin D fortification and ensure the levels remain adequate as egg production stabilizes or decreases.
o Studies indicate that the vitamin D content in eggs can remain relatively stable throughout the first year of laying if hens are kept on a consistent, fortified diet (Simsek et al., 2020).

3. Late-Laying Period (60+ weeks of age):
o As hens age, egg production tends to decrease. At this stage, it is still important to check the vitamin D content to ensure the continued effectiveness of supplementation. The level of vitamin D in eggs may decrease in older hens due to hormonal and metabolic changes.
o Some studies suggest that vitamin D fortification strategies might need adjustments of the fortified diet as hens age to maintain adequate egg vitamin D levels (Zhao et al., 2016).

Sampling and testing of fortified eggs .( Nick Fox et al .2009)
Sampling Eggs: Ensure eggs collected for testing represent the flock to capture an accurate average vitamin D level.
1. Sample Collection: Random samples of eggs should be selected from different batches at each stage (on the farm, during processing, and at retail). This ensures a representative analysis of the vitamin D content in the final product.
2. Sample size : To get a minimum number of necessary samples to meet the desired statistical constraints . With confidence interval 95% and Margin of Error 5, so if the Population Size around 80000 eggs per day in this case the estimated sample size should be around 383 eggs.
3. Testing Techniques:
o High-Performance Liquid Chromatography (HPLC): A common method to determine vitamin D3 levels in eggs.
o Liquid Chromatography-Mass Spectrometry (LC-MS): Provides a more detailed analysis, including measurement of
Khachik, F., Spangler, C., & Smith, J. C. (2004). etabolites like 25-hydroxyvitamin D3.

Packaging and storage conditions (Adam Clark et al. 2023) :
The stability of vitamin D in fortified eggs during storage is influenced by several factors, including packaging and storage conditions:
• carton packaging can help preserve vitamin D in eggs
1. protection from light.
2. Minimizing oxidation .
3. Temperature Regulation.
4. Humidity and Storage Quality.

• Recommendations for Carton Packaging :
1. Opaque and UV-Protective Materials: Use carton covers made from materials that block light (e.g., cardboard or UV-coated plastic).
2. Proper Storage Conditions: Combine effective packaging with optimal storage conditions (ambient or refrigerated, depending on duration) for best results.
3. Sealed Cartons for Long-Term Storage: If feasible, packaging that limits air exposure (e.g., sealed plastic-lined cartons) may further preserve vitamin D.

• Storage temperature :
Storing eggs at room temperature (20–25°C) for limited durations is generally suitable for preserving vitamin D levels, particularly in climates with moderate temperatures.
For prolonged storage, refrigeration might be preferred to prevent spoilage, even though it might slightly reduce vitamin D3 levels.

Recommendations for Vitamin D Fortified Eggs from Farm to Consumer:
To ensure consistent and effective vitamin D fortification in eggs throughout the supply chain, it is essential to establish monitoring system inside the farm that address all stages: on-farm production, processing, distribution, and retail., for maintaining optimal vitamin D levels in fortified eggs. (Mattila, P. H., et al. (2004).
1. The fortified eggs with vitamin D certification should be connected with the certification of the fortified dietary .
2. Monitoring the level of vitamin D must contain both fortified dietary traceability and routine sampling during laying period.
3. Fortified dietary must be systematic approved during sages of the laying production cycle .
4. To be convince that vitamin D in fortified eggs stable during the shelf life and in a suitable storage condition the stability study should be conduct during the shelf life of fortified eggs .
5. Testing and sampling procedure should be :
Begin sampling after hens are fully transitioned to a vitamin D- fortified diet, typically 4–6 weeks post-supplementation.

Routine Monitoring:
• Frequency: Every 2–4 weeks during:
o Peak production (24–30 weeks of age)
o Mid-laying period (40–50 weeks of age)
o Late laying (>60 weeks of age)

• Rationale: Regular checks are needed to verify that vitamin D content is consistent across production cycles and to account for seasonal variations or feed inconsistencies. Bie et al. (2019)
• suggest biweekly sampling during critical production periods.