Minimal effects of ultraviolet light supplementation on egg production, egg and bone quality, and health during early lay of laying hens

Animals and housing management

Sixteen-week-old ISA Brown layer (Gallus gallus domesticus) pullets (n = 252) reared in a deep litter commercial facility with curtained-sides were procured and transferred to the experimental indoor setting with environmentally controlled rooms. All the birds were from the same flock and no experimental interventions were applied during the rearing period. Once pullets were fully feathered, the curtains during rearing were opened to facilitate natural air ventilation into the house, enabling some exposure to daylight. However, the exact degree of this daylight exposure was unknown to the researchers given birds were commercially acquired from a distant location. Daily bird care, housing management, and feeding, during rearing were as per standard commercial protocols in accordance with the Model Code of Practice for the Welfare of Animals: Domestic Poultry (Primary Industries Standing Committee, 2002). Upon arrival, the pullets were orally dosed (via gavage) with medicinal treatment for mites (Exzolt®) as per the research facility policy which was repeated in the following week along with an oral de-wormer (CCD® Levamisole, CCD® Animal Health, Tamworth, NSW, Australia). Birds were distributed equally among eighteen floor pens (14 pullets/pen) within three rooms (six pens/room). Pens were separated by wire panelling with shade cloth for hens’ visual isolation and to prevent the scattering effect of the supplemental lights between pens. Each of the pens (3.2 m L × 1.75 m W) contained a single three-rung perch (1.07 m L × 64 cm W × 80 cm H), one rollaway nest box (34 cm L × 29 cm W × 24 cm H), one round feeder (40 cm H × 43.5 cm D × 1.36 m C), and two nipple drinkers. Resources either met or exceeded those set in the Model Code of Practice (Primary Industries Standing Committee, 2002) but no additional enrichments were provided in the experimental pens. Feed and water were provided ad libitum where a standard commercial grower crumble (Barastoc® pullet grower crumble, Melbourne, Australia) was fed on the first week of arrival, followed by commercial layer mash (Barastoc® Top Layer mash, Melbourne, Australia) for the remainder of the study period. Wood shavings were used as floor litter (5 cm depth). All birds were visually checked at least once daily to ensure any welfare issues were immediately detected and addressed.

Pens were artificially illuminated with poultry-specific LED white light bulbs (IP65 dimmable LED bulb, B-E27-10W-5K, Eco-Industrial Supplies, China) with a light intensity of 30 lux at birds’ eye height across the pens (Testboy TV 335 Digital LED Luxmeter, GmbH, Germany) when the birds were not close to or directly under the supplemental lights. The approximate hen stocking density was 2.5 hens/m². The hens were maintained on an ISA Brown specific lighting schedule (14L:10D at 16 weeks which was increased by 15 min each week until birds reached 24 weeks when the schedule was fixed at16L:8D for the remaining study period; lights on at 0500 h and off at 2100 h). The indoor temperature and humidity were mechanically controlled through an automatic ventilation system. Hens were leg-banded for unique identification to facilitate experimental measures. At 27 weeks, a subset of hens (four hens/pen; 24 hens/treatment; total 72) were sacrificed by cervical dislocation following an electrical stunning to assess the effects of light treatments on bone health. The euthanasia protocol was as per standards set by the Animal Ethics Committee of the University of New England. All remaining birds were rehomed at the end of the experiment.

Experimental design

On the day after bird arrival, pens were divided into three different lighting treatment groups (six pens/treatment): (i) UVO (420–724 nm): control LED-white light (IP65 dimmable LED bulb, B-E27-10W-5K, Eco-Industrial Supplies, China) with no UV spectral light supplementation; (ii) UVA (315–712 nm): LED-white light with supplementation of full-spectrum avian daylight (PureSun Compact Bird Lamp, E27-20W, Arcadia, Germany) containing UVA and UVB where 3 mm glass was used directly under the bulb to filter out the UVB spectrum; and (iii) UVA/B (288–714 nm): LED-white light with supplementation of full-spectrum Exo-Terra® (Rolf C. Hagen, Montreal, Canada) pet reptile bulbs (Reptile UVB200, 25W, PT2341) containing both UVA and UVB spectrums (see Figs. S1–S3 for spectrographs of the control and treatment bulbs). Location of treatment pens within each room were balanced across the three rooms. To account for increased lux from the supplemental bulbs in the treatment pens, the light intensity of the control pens was increased by adding an LED filament warm-white light (Edison Screw LED GLS Filament Globe, E27-4W, Mirabella, VIC, Australia) (Fig. S4). All supplemental bulbs were within Arcadia ceramic reflector clamp lamps E27 (200 mm, RARM160X) suspended 76 cm above the floor on one side of the pens. Thus, birds could choose to be located under the standard lighting or the illuminated area under the treatment light. Standing chicken eye height was approximately 40 cm (5 cm wood shavings) away from the light source but the chickens were closer if they stretched their necks (Fig. 1).

To facilitate UV irradiance exposure to the birds’ legs and feet (the greatest areas of UV radiation absorbance; Schutkowski et al., 2013), the perch was located on the treatment light side. If the hens were mounted on the perch, their legs were approximately 30–50 cm distance from the bulb depending on their movement on the different perch rungs. The intensity of light and UVB irradiance was greatest directly underneath the treatment light source which then gradually decreased due to scattering as distance from the bulb increased (Table 1). A timer was set in each pen to turn the treatment lights on/off in approximate accordance with the standard lighting schedule (best match possible based on the accuracy of the analogue timers). The light intensities were measured using a lux meter (Testboy TV 335 Digital LED Luxmeter, GmbH, Gerlingen, Germany), and the UV index was measured for the UVA/B treatment using a reptile UV index meter (Solarmeter Model 6.5 R UVI Reptile, Solarmeter Australia, Noosaville, QLD) at both floor level and hens’ eye height. The light spectrums and radiation irradiance were measured over an average of 10 readings using an Ocean Insight Flame-S-XR1 Spectroradiometer (200–1,025 nm, Quark Photonics, Melbourne, Australia) set with an integration time of 180,000 ms and integration range from 280 to 1,000 nm (Figs. S1–S4). The UV-filtering glass was cleaned with an alcohol wipe every 14 days to remove the dust.

Blood sampling, plasma and serum separation

At 20, 23, and 26 weeks, blood samples were taken in the morning between 0900 and 1200 h when the majority of the birds had laid their eggs. The same randomly selected subset of hens (five hens/pen; 30/treatment) was sampled each time (identified by numbered/colored leg bands). Three-experienced operators drained approximately 2–3 ml of blood from the wing veins of each hen using a 19-gauge needle. Approximately 1−1.5 ml of blood per hen was collected into a lithium-heparin plasma separation gel vacutainer (BD Vacutainer® PST Lithium Heparin Tubes, BD 368056, Franklin Lakes, NJ, USA) to separate plasma; while the rest of the blood was collected into another vacutainer (BD Vacutainer® Serum Tubes, BD367812, Franklin Lakes, NJ, USA) to separate serum. The vacutainers were immediately stored in an ice bath and shifted to the laboratory within 3 h after collection. On the same day, the plasma tubes were spun down in a bench-top centrifuge machine (Dynamica, Velocity 30R, Livingston, UK) for 15 min at 3,000 RPM. Then the supernatant portion of plasma was poured off into 2 ml microcentrifuge tubes using a plastic pipette and stored at −20 °C until further analysis. The serum tubes were kept at room temperature for 2–3 h to let the blood cells settle and then spun down in a bench-top centrifuge machine (Elmi® Ltd, CM-6MT, Riga, Latvia) for 15 min at 3,000 RPM. The supernatant portion of serum was then transferred into 2 ml microcentrifuge tubes using a plastic pipette and stored at −20 °C until further analysis.

Bone sampling

At 27 weeks, a total of 72 hens from the same subset of birds used for blood sampling (four hens/pen; 24 hens/treatment) were euthanized by cervical dislocation following an electrical stunning. The left tibia bone was excised and stored with flesh at −20 °C until further processing. Later, the bones were placed at room temperature overnight for thawing and then defleshed by hand using a scalpel before taking the final measures. Bones were wrapped in gauze-soaked saline to prevent drying until further procedures were conducted (see section Bone Health Assessments).

Egg production and egg quality measurements

Eggs from all pens were collected daily across the study period to measure hen-day egg production (%). Egg quality was measured twice at 21 and 25 weeks for all treatment groups. All eggs of a single day were collected in the morning, transferred to an egg quality testing laboratory, and measured on the same day (except eggshell weight and eggshell thickness) by a single experimenter. The eggs were not washed but were freed from litter debris before taking the measures. The order of quality testing within the treatment groups was not balanced across the day but all measures were taken within 4 h after egg collection. Any eggs that were significantly deformed were not included in the measurements. Egg length (mm) and width (mm) were measured using a Digital Vernier Caliper scale (Kincrome, Sydney, Australia) for calculating egg shape index (egg length divided by egg width); egg length was measured as the dimension between the poles, while the width was measured at the equator. Eggshell reflectivity (%) was then assessed using a shell reflectivity meter (Technical Services and Supplies, Dunnington, York, United Kingdom) with measurements taken in three places (equator, base and apex) and the average value used for the analyses.

Egg weight (g), eggshell breaking strength (Kgf), and other internal egg quality traits including albumen height (mm), yolk color, yolk height (mm), yolk diameter (mm), yolk index and Haugh unit were determined by using an egg multi-tester instrument (Nabel DET-6500, Kyoto, Japan). Afterward, eggshells were washed thoroughly in tap water to remove attached albumen from the shell surface and placed at room temperature for at least 48 h for air-drying before taking the eggshell weight (g) and eggshell thickness (mm). Eggshell weight (g) was measured on shells that had been washed and dried thoroughly, using an Adventurer™ Precision analytical balance (Model AX423, Ohaus®, NJ, United States). Eggshell (%) was measured as the percentage of total egg weight. Eggshell thickness (mm) was taken in three places (equator, base and apex) using a custom-built gauge, based on a Mitutoyo Dial Comparator Gauge (Model 2109A–10, Kawasaki, Japan) and the average value was used for the analyses.

Physiological blood measurements

Bone health assessments

Body weight and external health scoring

At 16, 20, 24, and 27 weeks, hens underwent body weight measurements using poultry-specific electronic hanging scales (BAT1; VEIT Electronics, Moravany, Czech Republic) and external welfare scoring based on the criteria in Tauson et al. (2005). This scoring system included feather loss across body parts (neck, chest, back, wing, vent, tail) and footpad lesions where a score of 1 indicated no damage, and a score of 4 the worst damage (i.e., bare skin, or bumble-foot for plumage and feet, respectively). All fresh or healing comb wounds were counted with toenail length measured in mm using a seamstress tape measure. Beaks were scored as 0, 1, or 2 indicating no, mild, or moderate/severe damage, respectively and keels were palpated and scored as 1, 2, or 3 indicating no, mild, or moderate/severe damage, respectively. Any other signs of injury or illness were noted such as swollen abdomen, eye infection, respiratory issues, and toe damage. The same trained individual did all the scoring across the study.

Data and statistical analyses

All data were analysed using JMP® 16.0 (SAS Institute, Cary, NC, United States) with α set at 0.05 and a trend was considered as P = 0.05 ≤ 0.10. Data were transformed where necessary to approach normality with studentized model residuals plotted for visual inspection of homoscedasticity. Eggs were collected on a daily basis when hens commenced laying at 17 weeks of age where the timing of sexual maturity was estimated for each treatment by determining the age when hen-day egg production (%) reached 50%. The hen-day egg production was determined on a weekly basis (the total number of eggs of a respective week per pen was pooled, then divided by the total number of hens per pen × 7 days × 100). The proportional data of hen-day egg production were logit transformed after subtracting a constant of 0.03 from all data to allow transformation of proportional values ‘1 or >1’ (100% or >100% production, n = 8 and 11 of 162, respectively) to approach normality. General linear mixed models (GLMMs) were applied to hen-day egg production with treatment and age (week) and their interaction as fixed effects, and room and pen as random effects. Both external and internal egg quality traits of the sampled eggs were measured twice at 21 (n = 222 eggs) and 25 (n = 237 eggs) weeks. Eggshell (%) and eggshell reflectivity (%) data were converted to proportions and logit transformed. Separate GLMMs were fitted to each egg quality variable (egg shape index, eggshell reflectivity (%), egg weight (g), eggshell weight (g), eggshell (%), eggshell breaking strength (Kgf), eggshell thickness (μm), albumen height (mm), yolk index, yolk color, Haugh unit) with treatment and age as fixed effects including their interaction; and pen and room included as random effects. Post hoc Student’s t-tests were applied to the least squares means where significant or trend differences were present, with the raw values presented in the tables and graphs.

To assess the effects of light treatments on blood physiological parameters, plasma Ca and P (n = 270: 5 hens × 18 pens × 3 age points), and serum 25(OH)D₃ (n = 191: 5 hens × 18 pens × 3 age points where 37, 29, and 13 samples were missing due to sample integrity at 20, 23 and 26 weeks, respectively) data were analysed by fitting separate GLMMs where treatment, age, and their interaction were fixed effects, and room, pen and hen ID were random effects. Both plasma Ca and P data did not require any transformation as the data were normally distributed while serum 25(OH)D₃ data were log transformed to approach normality. For bone quality analyses, the left tibia bone (n = 72: 4 hens × 18 pens) index was calculated using the following formula: Tibia index = (Tibia weight (g)/Body weight (g)) × 100. The percentage data of tibia ash, Ca, and P were converted to proportions and logit transformed. Tibia breaking strength and tibia ash content (g) did not require any transformation as the data were normally distributed. Data were analysed using separate GLMMs where treatment was a fixed effect, and room and pen were random effects. Post hoc Student’s t-tests were applied to the least squares means where significant or trend differences were present, with the raw values presented in the tables and graphs.

Growth and the health scoring data including the body weight, beak score, number of comb wounds, keel bone score, feather scores (neck, chest, back, wing, vent, tail), and toenail length at different age points (16, 20, 24, and 27 weeks) throughout the study period for individual hens from different light treatments were compiled (n = 1,008 data points/welfare parameter: 252 hens × 4 age points). The comb wound count data were square-root-transformed and the toenail length data were log transformed to improve normality. For the assessment of body weight, number of comb wounds, and toenail length, GLMMs were fitted with treatment and age and their interaction as fixed effects, and room, pen and hen ID as random effects. Post hoc Student’s t-tests were applied to the least squares means where significant differences were present, but the raw values are presented in the tables and graphs. An ordinal logistic regression was performed to analyse the beak scores and keel bone scores using treatment and age along with their interaction as fixed effects. There was no observed variation detected in the hens’ feather scores and footpad lesions irrespective of the light treatments and age during the study period (all hens had full plumage coverage and no footpad dermatitis) so these data were not analyzed.

Age at sexual maturity

This study showed that birds were sexually matured (50% hen-day egg production) at 19 weeks irrespective of the lighting treatments. However, hens under the UVA and UVA/B light treatments came into sexual maturity before the control birds (3 and 1 day earlier, respectively).

Egg production

There was a trend of increased hen-day egg production for the effect of UV lights relative to the control light (mean ± SEM: UVO: 74.15 ± 2.50%, UVA: 79.21 ± 2.50%, and UVA/B: 79.01 ± 2.50%; F_(2,13.04) = 3.55, P = 0.06), with post-hoc tests showing hen-day egg production under both UVA and UVA/B light treatments was higher than the control hens across the study period (P < 0.05; Fig. 2). As expected, the hen-day egg production significantly increased over age (F_(8,118.20) = 110.95, P < 0.0001; Fig. 2). There was no significant interaction between light treatment and age for hen-day egg production (F_(16,118.10) = 0.53, P = 0.93).

Egg quality

The effects of light treatment, age and their interactions on egg quality parameters are shown in Table 2. There were significant main effects of both treatment lights and age on eggshell reflectivity (treatment: P = 0.02; age: P < 0.0001) and egg yolk index (treatment: P = 0.01; age: P = 0.001). The eggshell reflectance increased for the eggs under both UVA and UVA/B light treatments and the egg yolk index decreased under UVA/B light, while both of the parameters were also affected by age with increasing reflectivity and decreasing yolk index across age. Parameters including egg weight, eggshell weight, eggshell percentage, eggshell breaking strength, eggshell thickness, and egg yolk color were not affected by light treatments (all P > 0.05) but these traits significantly increased across age (all P < 0.01, Table 2). Albumen height and Haugh unit significantly decreased across age (P < 0.0001) but were not affected by light treatment (both P > 0.05). There was only a trend for an interaction effect of light treatment and age on eggshell weight (P = 0.05, Table 2).

Bone quality

There was no significant effect of UV light treatments on the tibia bone index, tibia breaking strength, ash content, and ash percentage (all P > 0.05; Table 3). UV light also did not affect the tibia ash mineral concentrations of Ca and P (both P > 0.05; Table 3).

Physiological blood measurements

The UV light treatments did not have significant effects on the plasma Ca and P concentrations measured at different age points during the early laying phase (both P > 0.05; Table 4). However, there was an inconsistent increase in plasma Ca concentrations over age where the Ca concentrations were significantly higher in both the initial and final measurements at 20 and 26 weeks respectively compared to the second measure at 23 weeks (P = 0.04; Table 4). The plasma P concentrations were significantly lower in both the second and final measurements at 23 and 26 weeks respectively than in the initial measure at 20 weeks (P < 0.0001; Table 4). On the other hand, there was a trend for an effect of UV lights to increase serum 25(OH)D₃ over control lights (P = 0.09; Table 4), with post-hoc tests showing the UVA/B group had higher 25(OH)D₃ concentrations than the control group (P < 0.05; Fig. 3). There was no effect of age on serum 25(OH)D₃concentrations (P = 0.91; Table 4) and no interaction effects for light treatment and age on plasma Ca and P, and/or serum 25(OH)D₃ concentrations (all P > 0.05; Table 4).

Body weight and health welfare scoring

This study did not find a significant effect of light treatment on hens’ body weight across the study period (P = 0.19; Table 5). However, as the study period was during the hens’ grower stage and early lay, body weight significantly increased over age (P < 0.0001; Table 5). There was no significant interaction between light treatment and age on hens’ body weight (P = 0.68; Table 5). There was a significant interaction effect between light treatment and age on the number of comb wounds (F_(6,748) = 4.15, P = 0.0004; Fig. 4) with both UV light treatment hens showing a greater increase in comb wounds than control hens at 24 and 27 weeks. Toenail length showed a trend to decrease under control and UVA/B light treatments but significantly reduced over age and no interaction effect was observed (P = 0.09, P < 0.0001, and P = 0.27, respectively; Table 5). Light treatment and age affected beak score (P = 0.09 and P = 0.003, respectively; Table 5) but there was no significant interaction effect (P = 0.46; Table 5). The beak scores was higher in control and UVA/B hens than the UVA light treatment but increased after 20 weeks in all treatment groups. On the other hand, a significant interaction effect of light treatment and age was found on keel bone score (χ² = 14.33, df = 6, P = 0.03; Fig. 4). Keel bone damage score increased across age irrespective of light treatment, but a higher score was documented in UVA/B hens at 20 and 24 weeks than in UVA hens. The keel bone scores were all similar among hens from different light treatments at 27 weeks (Fig. 4). There were no skin lesions or other issues observed except toenail damage in 5 and 11 hens under UVO and UVA/B treatments, respectively. All hens irrespective of light treatment had good feather coverage and no footpad damage at each observation point across the study period.