Introduction

In recent decades, the egg layer-raising industry has undergone substantial transformation and shown extraordinary resilience in the face of unprecedented upheavals. The poultry sector’s advancement can largely be attributed to increased egg production, enhanced feed efficiency, and improved egg quality to meet market demands. As the annual egg demand rises by 1 million tons, with the current egg demand reaching 75 million tons, there is an urgent need for management strategies to maximize genetic potential. For example, to achieve a target egg mass of 20 kg per layer from 20 to 76 weeks of age, a minimum of 50 million layers will need to be recruited annually to the meet rising demands [1].

Although the global human population is predicted to rise to 9.7 billion by 2050, marking an increase of approximately one-third from the 2015 level, many countries still continue to have alarmingly high rates of different forms of malnutrition [2].

Europe consumes over 100 billion eggs annually, as eggs are prized for their excellent nutritional value and versatility as an ingredient across various cuisines. Furthermore, advancements in animal welfare standards and European legislation have resulted in the remarkable evolution of egg production techniques over the last 15 years [3].

Multiple studies have reported that hen egg output and quality are affected by both genetic and environmental variables [4]. Heritability calculations suggest that genetic variables account for 30–70% of phenotypic variation, while environmental factors account for the remaining 30–70. Thus, any alterations to egg characteristics necessitates the consideration of both genetic and environmental variables [5].

Light affects the mood, physical health, and productivity of chickens. By carefully regulating the spectral properties of LED light bulbs during brooding and raising periods, caged layer chickens could experience improvement in immunological efficiency, bone growth, and production effectiveness. These benefits are observed when considering a combination of light properties, such as sources, intensity, color, and photoperiod routine [6].

Increased lighting intensity may enhance bone growth in birds by facilitating increased exercise through activities such as leaping and flying [7]. Furthermore, a study observed reduced ionized Calcium (Ca) concentrations and higher bone formation indicators in Lohmann Brown Lite pullets raised under 50 lx for 8 weeks compared to those maintained under 10 lx. This suggests that calcium homeostasis and bone development in young Lohmann Brown Lite chickens may benefit from increased light intensity [8].

Unlike traditional chickens, modern hybrid hens lay more than 320 eggs annually. Chickens require a large amount of calcium (Ca) for egg shell formation, which constitutes 10% of the egg weight, with Ca accounting for 40% of its composition [9]. The eggshell serves as a barrier to prevent damage, safeguarding the eggs’ interior from the time of oviposition until consumption. However, in the later stages of the production cycle, as laying hens age (beyond 60 weeks or more), there is a noticeable drop in both egg and shell quality. This decline may be attributed to several factors, including the increase in egg size, decline in nutritional metabolism (particularly Ca), and an elevation in reproductive hormones (particularly estrogens) that occurs at the end of the production cycle [10, 11].

In birds, vitamin D is crucial for the metabolism of Ca and phosphorus (P). The chicken industry has long relied on vitamin D3 as it is a readily available form of the vitamin. Egg production is an extremely regulated process that depends on the availability of specific nutrients at specific periods during a 24-hour cycle. Calcium, an essential nutrient, constitutes 40% of eggshells. Moreover, Ca contributes to healthy eggshells and strong bones [12].

Supplemental vitamin D3 was most often detected in patients treated with cholecalciferol. Once absorbed by the intestine, cholecalciferol travels to the liver, where it undergoes 25-hydroxylation to form 25-hydroxycholecalciferol (25OHD3), after which it undergoes carbon 1-hydroxylation in the kidney to form 1,25-dihydroxycholecalciferol (1-25OHD3) [13].

Materials and method

Analysis of experimental diets

The diets used in this study were formulated according to the nutritional requirements of the “Lohmann Brown-Classic Layer,” which were either in line with or exceeded the recommendations of the NRC (1994) [16]. From 19 weeks of age onwards, the diet was supplemented in three phases: 19–50 weeks, 50–70 weeks, and > 70 weeks, with protein contents of 16.10%, 15.6%, and 14.8%, respectively. The diet contained 2700–2750 metabolized energy\kg. Each bird received a daily feed allowance of 115 g, as shown in (Table 1).

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Table 1. Ingredient composition of the experimental diets (% per fed basis, unless declared elsewhere).

https://doi.org/10.1371/journal.pone.0305099.t001

Experimental design

The experiment was designed to determine the differences between the control and treatment groups in terms of light durations and intensities, limestone ratio, and different doses of vitamins, as shown in Table 2.

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Table 2. The differences in feed consumption, light duration and intensity, limestone ratio, and different vitamin doses between the control and treatment groups.

https://doi.org/10.1371/journal.pone.0305099.t002

Results

Feed consumption and egg production rate

The daily average feed intake was unaffected by the varying limestone ratios given to the treatment group and by the different light intensity exposures, remaining consistent at 115 g for the control group and 112 g for the treatment group. However, despite this consistency, the treatment group exhibited a significantly lower feed consumption (p<0.01) compared to the control group.

A significant (p<0.05) interaction was observed between the level of limestone intake, ranging from the baseline level of 3.6% in the control and 3.7% in the treatment group (with the same light intensity and an additional duration of 5 minutes). Notably, an increase in the egg production rate of 0.9% was recorded until 29 weeks of age; however, this change was not significant (p<0.05). Subsequently, with regard to egg production, from to 30–39 weeks of age, the treatment group provided with a 3.8% limestone intake and higher light intensity (17 Lux/M²) and duration of exposure (8 minutes more), recorded a significant (p<0.05) 1.4% increase compared to the control group. From 40–49 weeks of age, the treatment group provided with limestone level L3 and higher light intensity (17 Lux/M²) and duration of exposure (8 minutes more) recorded a significant (p<0.01) 1.9% increase compared to the control group. From 50–59 weeks of age, the treatment group subjected to limestone level L4, and light levels Y4 and Z4 exhibited a significant (p<0.0001) 3% increase compared to the control group. From 60–69 weeks of age, the treatment group subjected to limestone level L5, and light levels Y5 and Z5 exhibited a significant (p<0.00000001) 5.8% increase in compared to the control group. In addition, from 70–79 weeks of age, the treatment group subjected to limestone level L6, and light levels Y6 and Z6 exhibited a significant (p<0.00000001) 8.6% increase compared to the control group. However, the highest increase was observed from 80–89 weeks of age, which were subjected to limestone level L7, and light levels Y7 and Z7, and (V6) showed a significant (p<0.000000003) 9.6% increase compared to the control group (Table 3).

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Table 3. The egg production, body weight, and other egg characteristics of the control and treatment groups.

https://doi.org/10.1371/journal.pone.0305099.t003

Mean of egg weight ratios

At 60–90 weeks, the treatment group exhibited significantly less (p<0.05) mean egg weights in eggs grades 53 g or lesser compared to the control group. Conversely, for eggs graded 63–73 g, the treatment group exhibited a higher mean egg weight compared to the control group (55.08 vs 51.37; p<0.05). However, the differences in the mean egg weights among the other egg groups were not significant (p˃0.05), as shown in Table 4.

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Table 4. Influence of calcium, light, and feed levels on egg weights ratios.

https://doi.org/10.1371/journal.pone.0305099.t004

Egg characteristics

At 75 weeks, the albumin weight, yolk weight, yolk diameter, shape index, and HU were not significantly different (p˃0.05) between the treatment and control groups. However, significant differences were observed in the treatment group compared to the control group for shell thickness (0.352 vs. 0.344; p<0.01) and shell weight (9.03 vs 8.79; p<0.0001), as shown in Table 5.

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Table 5. Influence of feed consumption, calcium, and light on egg characteristics.

https://doi.org/10.1371/journal.pone.0305099.t005

Broken and shell-less egg percentage

The percentages of broken and shell-less eggs were not significantly (p˃0.05) impacted at phase 1 between the treatment and control groups. However, the treatment group showed a significant decrease in phases 2 (p<0.00001) and 3 (p<0.000001) compared to the control group (0.715 vs. 0.946 and 0.86 vs 1.55, respectively). Additionally, shell strength was significantly (p<0.00001) increased across all the phases of treatment group compared to the control group (41.80 vs 38.40), as shown in Table 6.

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Table 6. Influence of feed consumption, calcium, and light on the percentages of broken and shell-less eggs.

https://doi.org/10.1371/journal.pone.0305099.t006

Discussion

The improvements in chicken performance is considered a major achievement in the poultry industry because as the hen reaches the end of its peak production ends, it lays fewer eggs and of poorer quality. This decline can be attributed to various factors, including a decrease in breeder hen vitellogenesis, lipogenesis, antioxidant, and immunological status, alongside potential issues such as poor management, improper feeding, genetic predisposition, or exposure to a toxic environment [17].

In the present study, feed restriction (112 g vs. 115 g) potentially affected feed consumption and egg production in the treatment group. These observations align with those of [18], which demonstrated that a feed limitation of 10% improved the laying performance, possibly due to enhanced maintenance processes regulated by body weight to prevent overeating [19, 20]. Notably, in this study, the feed reduction was less than 3% resulted in savings of over 55 tons of food at a price of 33,000 USD.

Supplementation of 3.8% limestone intake with a light intensity of 10–15 Lux/M² for a duration of 14 h + 5 min in the treatment group did not significantly (p<0.05) increase the egg production rate by 0.9% until 29 weeks of age. However, from week 30–39, the treatment group with limestone intake of 3.9% and light intensity 17 Lux/M² and duration of 14 h + 8 min, combined with two doses of vitamin AD3E, exhibited a significant (p<0.05) 1.4% increase compared to the control group. By the end of the experiment, the treatment groups with the limestone intake parameter L7, and light conditions Y7 and Z7 recorded a significance (p<0.000000003) 9.6% increase in egg production compared to the control group that was reared according to the guide’s specifications.

The results obtained in this study represent a pioneering achievement in the field, as they demonstrate the combined effects of limestone, light, and vitamin supplementation on egg production. Interestingly, previous research has indicated varying outcomes regarding the influence of calcium intake on egg production in chickens. For instance, some studies have shown that increasing Ca intake from 3.68 to 4.26 g/d (3.22 to 3.83% of the diet) did not affect egg production in chickens aged 55–70 weeks [21]. Similarly, another study reported that in chickens aged between 24–27 weeks, neither egg production nor egg weight was influenced by the increase in Ca intake from 3.58 to 4.35 g/d (3.3 to 4.1% of the diet [22]. Moreover, a study that examined chickens aged 38–62 weeks reported that increasing the Ca component of the feed from 3.85 to 4.40 g/d (3.5 to 4.0% of the diet) had no effect on the egg production, egg weight, or FCR [23].

Conversely, in 21-week-old W36-Hyline hens, an elevation in Ca intake (from 2.1 to 4.2 g of Ca/b/d) at a fixed dietary dose of available phosphorous (P) (0.40%) was observed to boost egg production characteristics, as reported by [24]. Similarly, increases in both egg production and egg quality characteristics were observed when the Ca:P ratio was maintained at 12:1 (with a Ca consumption of 4.1 to 4.6 g of Ca/b/d). Additionally, both [25, 26] reported that giving both young and grown chickens a Ca supply in the form of large pieces (0.8–2 mm) of limestone boosted both egg weight and production. Consequently, the external and internal quality characteristics of eggs produced before and after the age of 39 weeks exhibit significant changes [27].

In the present study, the observed increase in egg production may be attributed, in part, to the varying light intensities applied to hens from ranging from 10 Lux/M² to Y7. These results align with those of [28], which suggest that light properties play a role in managing egg quality. Moreover, light sources [29], light strength [30], and the homogeneity of light distribution [31] have been reported to affect the laying productivity of chickens.

Consistently, the inclusion of vitamin supplementation in the drinking water of the control group, in addition to rations, was sound to be crucial in maintaining with the productivity of hens. These findings align with those of [32], which observed significant increases in egg production, egg mass, fertility, and hatchability of eggs with the inclusion of vitamin A supplementation in the diet of layer. Similarly [33], the reported that the addition of vitamin E to the diet improved both egg production and quality. Additionally [34], attributed recorded increases in both egg production and quality to the early and long-term utilization of vitamin D.

Notably, in this study, the addition of different doses of vitamins across all experimental periods resulted in increased egg production.

There was no significant (p>0.05) difference in body weight between the groups from 20–69 weeks, likely due to the feed restriction of less than 3% in the treatment group. However, the differences in weight were obvious from 70–79 and 80–89 weeks, with the treatment group recording significantly lower weights (p<0.01 and p<0.001, respectively). This could be attributed to the cumulative effect of reduced feeding over long periods, preventing overweight conditions and maintaining correct body weight [19]. Moreover, the FCR/egg ratio remained relatively unchanged from to 20–29 weeks, but a significant difference (p<0.0000001) emerged from 30–90 to weeks in the treatment group, indicating conserved high egg production despite lower feed consumption. These results align with those of [18, 35, 36], which demonstrated that a feed limitation of 10% improved laying performance, by aiding in the preservation of proper body weight and preventing overeating.

These results were in partial disagreement with [18, 36], as they advocated for minimizing feed consumption by more than 20 or 30 percent of ad libitum, whereas this study minimized feed consumption by less than 3%.

Despite the increases in egg weight and egg mass, no significant (p>0.05) differences were found between the two groups from 20–29 weeks. However, from 30–89 weeks, the treatment group showed gradual and significant increases, attributed to the combination of all the treatment parameters (many doses of limestone, many types of light intensity, and many doses of vitamins), consistent with those reported by [25, 26].

The treatment group recorded a significantly (p<0.05) lower mean egg weight than the control group from weeks 60–90 in the category of eggs weighing 53 g and lesser, while it was higher in the category of eggs weighing 63–73 g. However, the differences among the other categories were not significant. These findings indicate a favourable profit margin and the most desirable egg size (63–73 g) for human consumption. This aligns with the results reported by [37–39], which demonstrated that hens reared in furnished cages with different grades of limestone and Vitamin D under an intermittent light regime had better eggshell quality and egg weight.

Although the albumin weight, yolk weight, yolk diameter, and HU were higher in the treatment group than those in the control group, the increases were not significant at 75 weeks. However, the shell weight and shell thickness of the treatment group showed significantly higher (p<0.0001 and p<0.01) differences. The findings align with those of [40], which demonstrated that enhanced scotophase increased the Ca and P content in the eggshell and improved eggshell hardness. Similarly [25, 41], indicated that dietary Ca and P and levels influenced eggshell quality throughout the laying phase to guarantee eggshell quality in the final third of the production period. However, when brown-egg-laying chickens were fed [42] a Ca intake of more than 3.4 g/d, eggshell deformation was not affected. The Ca was delivered in the form of limestone. Therefore, variations in strain, age, egg output, and nutritional configurations of the diets may account for the inconsistency among studies regarding Ca requirements and eggshell quality.

The increase in the proportion of broken and shell-less eggs between the two groups was not statistically significant throughout the first phase of the study. However, significant improvement was observed in the treatment group over the course of phases 2 and 3, with the latter showing an increase in outcomes with a P-value less than 0.000001. These results seem to align with [12, 25], which demonstrated that an increase in Ca consumption enhanced shell weight, thickness, and density, and decreased the proportion of broken and shell-less eggs. Notably, the last stage of the egg production exhibited the greatest increase in shell weight and density.

Since egg production in laying hens is directly related to the nutrients they receive in their feed, changes in diet formulation may help the poultry industry achieve its ultimate aims. Producers prioritize diet quality and composition because feed accounts for 70–80% of the total cost of egg production [43]. Least-cost linear programming can be utilized to create a diet that is inexpensive while satisfying the birds’ nutritional requirement. While individual businesses and companies may have differing priorities, most commercial enterprises strive to maximize revenue by optimizing their inputs and outputs. The formulation of poultry diets and decision-making processes can benefit from the rapid development of big data and communication technologies [44]. Weekly live performance evaluations, the total egg yield per house, and the treatment expenses for each house were used for financial evaluation. The expected profits from selling eggs served as the basis for calculating the gross margin (30-dozen-egg package). As all treatments followed the same management and feeding protocols and had access to the same housing and infrastructure, there was no difference in labor allocation. Consequently, the economic analysis excluded such expenses.

Conclusion

The innovative nutritional protocol outlined in this study decreased the final feed consumption of the treatment group by more than 55 tons, resulting in saving equivalent to 33,000 USD. Moreover, the increase in egg production within the treatment group amounted to more than 1,425,408 eggs compared to the control group and the strain’s standard guidelines over 490 days of production, equivalent to 142,541 USD. At the same time, there was a significant decrease in light color and shell-less eggs in the treatment group, contributing as addition 5000 USD in profit.

Finally, the treatment group yielded a total revenue of 180,541 USD, surpassing that of the control group (with profit exceeding the control group’s baseline profit).

This study represents a pioneering effort worldwide, conducted in two houses under field circumstances, each housing 75,000 layers for 89 weeks. These results signify significant achievements in the poultry industry, demonstrating excellent revenue and advancements.

Acknowledgments

The author would like to thank the Middle East Agricultural Company Ltd. for their assistance in completing the project. This study was performed at the College of Veterinary Medicine, University of Kufa. The authors received no funding for this study.