In this study, we evaluated the impact of moderate and high dose dietary supplementation of astaxanthin on production performance, quality of eggs, and health status of laying hens. The experiment involved 480 laying hens, divided into four groups of eight replicates. The different groups named A1, A2, A3, and A4 were allocated the same diet supplemented with Haematococcus pluvialis powder to provide 0, 21.3, 42.6, and 213.4 mg of astaxanthin per kilogram of feed, respectively. One-way ANOVA and linear and quadratic regression analysis were used to assess the differences between the groups. The results showed that the production performance of laying hens and the physical quality of eggs did not significantly differ between the groups (p > 0.05). Astaxanthin distribution in tissues was typical per bird, whereas the egg yolk coloration and astaxanthin concentration increased with the supplementation dose (p < 0.001). However, there was a decrease in concentration and coloration efficacy of astaxanthin at high dose supplementation (213.4 mg/kg) compared to moderate doses (21.3 and 42.6 mg/kg). Blood biochemical tests showed some discrepancies that were not ascribed to the effect of diets, and the increase in liver weight in the A4 group compared to others was equated with an adaptation of laying hens to the high dose supplementation. Astaxanthin improved superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and diminished malondialdehyde (MDA) content in both liver and serum; meanwhile, the activities of SOD and GSH-Px in serum were similar between the moderate doses and high dose supplementation. Additionally, astaxanthin alleviated interleukin 2, 4, and 6 (IL-2, IL-4, and IL-6, respectively) in serum, showing the best effect in A3 and A4 groups. Besides, immunoglobulin G and M (IgG and IgM), as well as tumor necrosis factor-alpha and beta (TNF-α and TNF-β), were not much affected. It was concluded that although astaxanthin has no obvious adverse effect on the performance and health status of laying hens, it may not be valuable for egg fortification and health status improvement of laying hens at high dose supplementation. The high dose astaxanthin supplementation up to 213.4 mg/kg in the diet might be avoided.

Long-term and graded dose of astaxanthin supplementation in laying hen's diet was assessed for egg fortification. Five groups of laying hens with 8 replications each were fed for 24 wk with diet supplemented astaxanthin at 0 mg/kg (control), 7.1 mg/kg, 14.2 mg/kg, 21.3 mg/kg, and 42.6 mg/kg ( Basal, A7, A14, A21, and A42, respectively). The performance of laying hens, egg quality, astaxanthin concentration as well as conversion efficiency and geometric isomers proportion in yolks were assessed on wk 8 and 24. One-way analysis of variance ( ANOVA ) and linear and quadratic regression analyses were used to evaluate the dose effect. In parallel, the Student's t test compared the values between wk 8 and wk 24 of test within a group. Overall, the results revealed that neither production performance nor egg physical quality was affected by astaxanthin dose level and feeding duration. Following the supplementation dose, the redness of yolks ( a* ) increased ( P < 0.001). But, the a* score in A42 (23.48) was just 3-folds the a* score in A7 (8.89). Concentration of astaxanthin in eggs was dose-level dependent showing a linear relationship ( P < 0.001) with a slight declination observed in all groups on wk 24 compared to wk 8. The deposition rate of astaxanthin into egg yolk was higher in A21 and A42. The proportion of geometric isomers in egg yolk were not affected by the feeding duration. As the supplementation dose increased, all-trans isomer proportion gradually decreased in the egg yolk, while 13-cis isomer proportion rose. It was concluded that astaxanthin is an efficient carotenoid for egg fortification, which can be supplemented in diet up to 42.6 mg/kg for 24 wk without compromising the performance of laying hens or physical quality of eggs. This appreciably affects the egg yolk color and confers a better accumulation of total astaxanthin and cis isomers into eggs as the supplementation dose increases.

Summary Production of the high‐value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing β‐carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed‐specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2–16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10–14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long‐term storage of astaxanthin‐rich maize are at 4 °C in the dark. This study shows that co‐expressing of functional genes driven by seed‐specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost‐effective plant factory for reliably producing astaxanthin.