Significant difference on Yellowness (b) The Intra Calferol supplementation was clearly observed in the yellowness of the egg yolk. Although at the start of the trial (week 69), the control group showed a significantly ‘yellower’ yolk, a shift was seen by the end of the trial period at week 81 and the egg yolks of the Intra Calferol supplemented group had significantly yellower yolks (figure 1).

Figure 1: Yolk colour yellowness of control group compared to Intra Calferol supplemented group. Datapoints with an asterisk denotes significant differences.

Improved redness (a)

The results in figure 2 demonstrated that when it came to the yolk redness, even though the treatment group yolks were significantly less red at the start of the trial (week 69), the supplementation of Intra Calferol resulted in an improved redness and thus was eventually similar to the control group in terms of redness. In short, supplementation of Intra Calferol helped the treatment group ‘catch-up’ to the control group.

Figure 2: Yolk colour redness of control group compared to Intra Calferol supplemented group. Datapoints with an asterisk denotes significant differences.

Same Lightness (L)

The egg yolk lightness (L) for both groups showed a similar trend of increase over time with no statistical significance. However, since egg yolk redness (a) and yellowness (b) were significantly improved while lightness was similar between both groups, the improvement of the egg yolk visual quality was evaluated further based on the Delta-E gap.

Intra Calferol has closed the Delta E-gap

The Delta E quantifies the difference between two colours. Delta E is calculated on a scale from 0 to 100 (see below table 1). A low Delta E (< 1) indicates identical colouring, while a high Delta E level of 100 indicates exactly opposite

colours.

table 1: L*a*b* colour system to calculate Delta E is based on the standard formulation: Δ E*ab = ((Δ L*)2 + (Δ a*)2 + (Δ b*)2)0,5

The Delta E value at T0 was 2,97 (perceptible at a glance). At T1 it already was closed to 1,82 and even further on T2 to 1,24, a difference hardly not perceptible. Intra Calferol group has clearly caught up, clearly closing the gap.

β-carotene concentrations confirm the better egg yolk colouring

Additionally to the Delta E values, the β-carotene concentration in the yolk was measured. Randomly 30 collected eggs per group were separately measured at week 81 (T2) and independently measured by the lab of a leading egg producer for β-carotene. The improvement in egg yolk colour after supplementation with Intra Calferol could be related to the average increase of 13% in the β-carotene deposition. β-carotene is the organic pigment responsible for red-orange colouring and higher concentration of this pigment in the yolks of Intra Calferol supplemented eggs lead to a more intense, golden colour of the yolk which also reflected in the L*a*b* colour value.

figure 3: Mean beta-carotene concentration of 30 randomly sampled egg yolks

In conclusion, older laying hens supplemented with Intra Calferol produced better quality eggs with more golden yolks with higher beta-carotene level. This was most likely attributed to the improved health status of the flock therefore facilitating more pigment absorption via the gastrointestinal tract and better pigment deposition in the yolk via the reproductive system.