Genotype-dependent impact of dietary vitamin D3 on laying hens

ABSTRACT Vitamin D3 has an integral part in calcium and phosphorus homoeostasis, which in turn plays a key role in egg production of hens. The present study aimed to investigate whether an additional vitamin D3 supplementation improves the laying performance and egg quality of hens according to their genetic potential. For this purpose, four layer lines (low performing: R11 and L68; high performing: WLA and BLA) supplemented either with 300 or 3000 IU vitamin D3 per kg feed were compared concerning serum 25-hydroxyvitamin D3 (25-OHD3), calcium, phosphorus and alkaline phosphatase (ALP), laying performance and egg quality. The higher supplementation of vitamin D3 increased 25-OHD3 serum concentrations in all genotypes, except for R11 and WLA hens in week 49, and also elevated vitamin D3 and 25-OHD3 content in the egg yolk (p < 0.05). In week 29, 3000 IU vitamin D3 decreased pooled least squares means (LSMeans) of serum calcium concentrations considering all genotypes and increased the ALP concentrations in BLA hens (p < 0.05). Considering the whole experimental period daily egg mass of R11 hens was increased by an additional vitamin D3 supplementation (p < 0.001). Regarding all genotypes and the whole experimental period the pooled LSMeans of breaking strength of eggs from hens fed 3000 IU vitamin D3 were higher than those of hens fed 300 IU (p = 0.044). In conclusion, present results give evidence that the higher vitamin D3 supplementation might have genotype-dependently beneficial effects on calcium and phosphorus homoeostasis of hens, which might improve feed efficiency in the early laying period and promote the persistence of the laying period irrespectively of genotype. The increase of serum 25-OHD3 by the higher vitamin D supplementation supported the higher transfer of vitamin D in the egg yolk and improved genotype-dependently the breaking strength of the eggshell.


Introduction
Vitamin D 3 can be synthesised in the skin during UV exposure or ingested as vitamin D 2 or D 3 from dietary sources or supplements (Soares et al. 1995;de Matos 2008;Proszkowiec-Weglarz and Angel 2013). Birds that are kept indoors rely on a dietary vitamin D supplementation (Edwards et al. 1994;de Matos 2008). In the avian nutrition vitamin D 3, which has a markedly higher biological activity in birds, is the commonly used vitamin D isoform (de Matos 2008;Proszkowiec-Weglarz and Angel 2013). Vitamin D 3 undergo two hydroxylation steps which initially lead to the formation of 25-hydroxyvitamin D 3 (25-OHD 3 ) and subsequently to the active form 1,25-dihydroxy vitamin D 3 or 24,25-dihydroxy D 3 (de Matos 2008;Proszkowiec-Weglarz and Angel 2013). The vitamin D hormone plays a key role in the intestinal calcium and phosphorus homoeostasis and utilisation which are crucial for bone and eggshell mineralisation and on the other hand, the activation of vitamin D 3 depends on plasma calcium concentration (de Matos 2008;Adedokun and Adeola 2013;Proszkowiec-Weglarz and Angel 2013). Factors influencing the vitamin D requirements are for example the species, age, breeding status, egg production, growth, fertility or hatchability as well as the calcium and phosphorus levels in the diet (de Matos 2008).
In previous studies, it was shown that an additional vitamin D 3 supplementation could influence the laying performance of hens by increasing the daily egg production or the egg weight (Atencio et al. 2006;Browning and Cowieson 2015). Besides the laying performance of the hens also the egg quality plays an important role. The eggshell quality representing an integral part of the egg quality influences the percentage of broken eggs. Thus, the eggshell quality has a high economic priority and depends on breeds, lines and families of laying hens as well as the age of hens (Ketta and Tumová 2016). Additionally, dietary supplementation with vitamin D 3 or 25-OHD 3 also shows beneficial effects on the eggshell quality (Soares et al. 1995).
Given the specific role of vitamin D for calcium homoeostasis and in turn of eggshell stability, we hypothesised, that the current recommendation of 300 IU vitamin D 3 /kg diet (GfE 1999) is insufficient for modern laying hybrids which are characterised by a high laying performance. We assume that these hens might benefit from higher vitamin D 3 doses. According to EC No 1831/2003(2003 the maximum permissible vitamin D 3 content in laying hen feed is 3000 IU vitamin D 3 /kg diet. Moreover, hens laying brown-coloured-coloured eggs are characterised by a calcium and bone metabolism which is different from those laying hens that produce whitecoloured eggs (Habig and Distel 2013). Thus, it was further hypothesised that phylogenetically divergent layer lines would respond differently to low and high doses of vitamin D 3 . The current study investigated the effects of 300 IU vs. 3000 IU vitamin D 3 /kg feed in phylogenetically divergent layer lines with marked differences in laying performance. Therefore, the four layer-line model at first reported by Lieboldt et al. (2015) was used to investigate the interactions between genetically determined laying performance and vitamin D 3 supply.
Summarising, the present study aimed to investigate the influences of a vitamin D 3 supplementation of 300 IU vs. a vitamin D 3 supplementation of 3000 IU on laying performance, egg quality and calcium-phosphorus homoeostasis in various genotypes of laying hens differing in performing type and phylogeny according to brown and white layer lines at different ages. It was hypothesised that a higher vitamin D 3 supplementation improves the laying performance and also the eggshell mineralisation in the hens and might reduce the genotype dependent differences.

Materials and methods
The current study is a companion study of an interconnected research project dealing with the effects of phylogeny and performance divergence on the adaptability of layer lines (Jansen et al. 2020). The present study was performed at the experimental station of the Institute of Farm Animal Genetics, Friedrich Loeffler Institute (FLI), Federal Research Institute of Animal Health in Neustadt, Germany. The housing conditions of the animals and experimental procedures described in this study were in accordance with the guidelines of the German animal protection law and were approved by the Lower Saxony State Office for Consumer Protection and Food Safety, LAVES, Germany (registration number: AZ 33.19 -42502-04-15/1988).

Experimental diets and design
Laying hens of four genotypes (72 hens per genotype; 36 hens per feeding group) were supplemented either with 300 IU vitamin D 3 (c, recommendation according to GfE (1999)) or +, 3000 IU (the maximum permissible vitamin D 3 content according to EC No 1831/2003(2003) from week 18 of life (Supplemental Table S1). The four genotypes included two low performing genotypes (L68 and R11 hens) and two high performing genotypes (BLA and WLA hens) differing in their phylogeny (brown layer lines (L68 and BLA) vs. white layer lines (R11 and WLA)). The basal diets were formulated according to the requirements of the different performance periods and the recommendations of the National Research Council (NRC 1994) and the Society of Nutrition Physiology (GfE 1999) (Table 1). Feed and water were available ad libitum by feeding troughs and nipple drinkers. The hens were equipped with win-tags and reared in groups in conventional floor-range pens until starting the laying trial. Within this period the light and temperature program followed usual specifications of rearing layer chickens. During rearing birds were vaccinated against Marek´s and Newcastle Disease and Infectious Bronchitis.

Laying trial
After rearing at week 17, birds were placed in random order into a layer facility with a three-floor cage system including single cages (50 cm × 46 cm × 43 cm) containing a perch, a nipple drinker and a feeding trough. Cages contained no litter material. The birds were housed at a temperature of 18°C without artificial ultraviolet B light exposure. From week 17, the daily lighting period was increased by half an hour per week up to a constant illumination period of 14 h from week 23 on. The feed intake and the number of laid eggs were recorded weekly. In 4-week intervals the eggshell weight including inner egg membrane, eggshell thickness and strength were determined.

Balance trial
To study the apparent nutrient retention the laying hen feed was blended with Sipernat Ⓡ (Evonik Industries, Essen, Germany) as indigestible marker. In week 29 and 49, the excrements of 8 animals per group were collected over three consecutive days and pooled per animal. The collected excrements were stored at −20°C until further analyses. On the last sampling day of both balance periods and at the end of the experiment (week 69) blood samples of the observed hens were taken from V. ulnaris s. basilica and collected in conventional serum tubes.
During the sampling period in week 29 also the laid eggs from the 8 birds (corresponding to birds from which excrements were collected) were collected and the oviposition time was noted (time interval 1: before or at 7:30 am; 2: between 7.30 am and 12:00 pm, 3: after 12:00 pm). The weights of egg yolk, egg white, and eggshell including inner shell membrane were determined. The thickness and breaking strength of the eggshell were measured. The yolk colour was determined according to a Roche-fan (1-white to 15-intense orange). Thereafter, the yolk was frozen at −20°C until further analyses.

Analyses of feed and excreta
Feed and excreta samples were analysed according to methods of the Association of German Agricultural Analysis and Research Centres (VDLUFA). DM of feed and excreta  were analysed by methods of Naumann and Bassler (2006). Nitrogen was determined by the combustion-method according to Dumas (Naumann and Bassler 2006). Methods of VDLUFA 8.1 and 8.2 were used to analyse crude ash and HCL-insoluble ash, respectively. The mineral concentrations of calcium and phosphorus were analysed with optical emission spectrometry according to method number 10.8.2.

Vitamin D analyses in egg yolk
Freeze-dried egg yolk samples were analysed for their vitamin D 3 and 25-OHD 3 content by high-performance liquid chromatography tandem-mass spectrometry (HPLC-MS /MS) as described by Kühn et al. (2019). In brief, samples were spiked with deuterated internal standards. After saponification under alkaline conditions, samples were extracted with n-hexane, washed with ultrapure water and subjected to normal phase HPLC (1100 Series, Agilent Technologies, Waldbronn, Germany) for further purification. Based on the specific retention times, vitamin D 3 and 25-OHD 3 were collected in separate fractions. Both fractions were derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione and injected into the HPLC-MS/MS (1200 Series, Agilent Technologies; QTRAP 5500, Sciex, Darmstadt, Germany). Ionisation was achieved by positive electrospray. Data were recorded in the multiple-reaction monitoring mode with the following ion transitions: vitamin D 3 : 560 > 298, deuterated vitamin D 3 : 563 > 301, 25-OHD 3 : 576 > 298, deuterated 25-OHD 3 : 582 > 298. In case of 25-OHD 3 concentrations in egg yolk below the quantification limit (<16 ng/g DM), a concentration of half of the quantification limit was used for statistical calculations.

Calculations and statistical analyses
The beginning of the laying period and therefore the laying maturity were defined as the time point reaching a laying intensity of at least 50% per week for two consecutive weeks.
In contrast, the end of the laying period was defined as the time point when the laying intensity dropped below 50% per week for at least three consecutive weeks. If the laying intensity did not drop below the reference point before the end of the trial (week 69), an end of the laying period in week 70 was supposed. Five birds did never reach a laying intensity of at least 50% because of premature exclusion from the trial. They were not considered for the analyses of parameters in the laying period.
The nutrient retention was calculated by the following equation: The calcium and phosphorus intake was calculated according to analysed calcium and phosphorus concentrations in feed related to the real feed intake of each bird. The feed conversion ratio is defined as the quotient between the daily feed intake (g) and the daily egg mass (g). The eggshell density was calculated by dividing the eggshell mass (g) by the eggshell thickness (mm).
The SAS Enterprise Guide 8.1 for Windows (SAS Institute Inc., Cary, NC) was used to evaluate the present results. The MIXED procedure was used to analyse the apparent nutrient retention, blood traits, traits of laying performance and eggshell traits measured during the laying trail. The model included the fixed effects "week of life", "genotype" (L68; R11; BLA; WLA) and "vitamin D supplementation" (300 IU vitamin D 3 vs. 3000 IU vitamin D 3 ). Repeated measurements of the birds were considered using the REPEATED statement. In case of blood traits, the SLICE statement of the MIXED procedure was used to conduct partitioned analyses for the LSMeans for interactions. In case of egg quality parameters and vitamin D content in egg yolk determined during the balance trail in week 29 as well as the analyses of the laying period or of the ELP ratio, the model of the MIXED procedure included the fixed effects "genotype" and "vitamin D supplementation". To analyse the effects of oviposition time on eggshell thickness or strength the time interval (1-4) of oviposition was also considered as a fixed factor. Least squares means (LSMeans) and their standard errors were calculated for each fixed effect, and all pairwise differences in LSMeans were tested with the Tukey-Kramer procedure. Effects and differences were defined as significant if p < 0.05. The SAS Enterprise Guide 6.1 was also used to estimate Pearson correlation coefficient; r. Correlations were assessed as significant at p < 0.05.

Feed analyses and apparent nutrient retention
The calcium content in laying hen feed was unintentionally higher in the diet containing 300 IU vitamin D 3 (50.41 g/kg DM) compared to the diet containing 3000 IU vitamin D 3 (44.12 g/kg DM). In contrast, phosphorus content was slightly higher in the diet containing 3000 IU vitamin D 3 (8.52 g/kg DM) compared to the diet containing lower amounts of vitamin D 3 (7.96 g/kg DM). Therefore, the calcium: phosphorus ratio was 6.35 for the 300 IU vitamin D 3 and 5.18 for the 3000 IU vitamin D 3 .
In general, pooled LSMeans of organic matter, crude ash, nitrogen, phosphorus and calcium retention were lower at week 29 compared to week 49 of life (p < 0.001) ( Table 2). The nitrogen retention was lower in L68c hens compared to WLAc hens in week 29 of life. Crude ash and nitrogen retention were lower in L68+ and R11+ hens compared to BLA+ and WLA+ in week 29 of life (p < 0.05) except for nitrogen retention of R11+ hens. The phosphorus retention was lower in BLAc hens compared to WLAc hens in week 29 of life (p < 0.05). In week 49 of life the organic matter, crude ash and calcium retention was lower in L68c hens compared to the high performing hens supplemented with 300 IU vitamin D 3 except for the organic matter of BLAc hens. In week 49, a 3000 IU vitamin D 3 supplementation decreased the phosphorus retention in all genotypes (p < 0.001).

Concentrations of 25-OHD 3 , calcium and phosphorus in serum
The 25-OHD 3 concentrations in serum of control groups did not differ between genotypes (p > 0.05) (Figure 1(a)). The higher supplementation of vitamin D 3 increased 25-OHD 3 serum concentrations in all genotypes and at all sampling time points (p < 0.001), except for white layer lines (R11; WLA) in week 49 showing only a numerical increase (p > 0.05). In week 49 and 69, the 25-OHD 3 concentrations in serum were higher in BLA+ compared to WLA+ hens (p < 0.01). In week 69, the 25-OHD 3 serum concentrations were also higher in the BLA+ group compared to the R11+ group (p = 0.002). Serum concentrations of calcium and phosphorus were affected in an age dependent manner (p < 0.05), showing highest values in week 29 and lowest in week 49 (Figure 1(b,c)). In week 29, pooled LSMeans of serum calcium concentrations were lower in the group fed 3000 IU vitamin D 3 compared to pooled LSMeans of birds supplemented with 300 IU (p = 0.045). There were no differences between the layer lines at similar time points.
A significant feed-by-genotype-by-week interaction was detected in case of the serum activity of ALP (p = 0.049) (Figure 1(d)). In WLAc and BLAc hens the ALP activity was increased in week 29 compared to week 49 and 69 (p < 0.001). Furthermore, in week 29 WLAc hens showed higher serum ALP activity compared to R11c and L68c hens (p < 0.05). Supplementation of 3000 IU vitamin D 3 decreased the ALP activity in BLA hens compared to hens fed 300 IU in week 29 (p = 0.018). In week 49, WLA+ hens showed higher ALP activity compared to L68+ hens (p = 0.015).

Vitamin D 3 and 25-OHD 3 concentrations in egg yolk
The vitamin D 3 and 25-OHD 3 content in egg yolk was similar in all control groups but tended to be slightly higher in the BLAc group compared to the WLAc group (p = 0.058) (Figure 2(a,b)). The higher vitamin D 3 supplementation increased the vitamin D 3 and 25-OHD 3 content in the egg yolk of all genotypes (p < 0.05). The BLA+ group showed significantly higher vitamin D 3 content in egg yolk compared to the other genotypes with similar vitamin D supplementation (p < 0.05). The 25-OHD 3 content in egg yolk was also higher in the BLA+ group compared to the R11+ and the WLA+ group (p < 0.05).

Performance
While the laying maturity was reached earlier in the high performing genotypes compared to the low performing genotypes (p < 0.001) showing also a difference between latter ones (p < 0.001) (Figure 3(a)), the end of the laying period was similar between all genotypes. In turn, the laying period of high performing genotypes lasted longer than that of low performing birds (p < 0.01) (Figure 3(b)). The vitamin D 3 supplementation had no influence on the beginning, end or duration of the laying period (p > 0.05). There was a significant week-by-genotype-by-vitamin D 3 -interaction in case of egg production rate (p < 0.05) (Figure 3(c)). In all genotypes the egg production rate increased until week 28. While the production rate remained on a high level in BLA and WLA hens irrespectively of the vitamin D supplementation the egg production rate of low performing hens seemed to decrease steadily after week 32 except for R11 hens receiving 3000 IU vitamin D 3. These hens show no difference in the egg production rate compared to BLA+ hens until week 64 of life. p week x genotype x vitamin D = 0.674; (d) alkaline phosphatase, ***Asterisks mark significant differences between feeding groups of similar genotypes (p < 0.001). a-c; A-C different superscripts mark significant differences between genotypes in the control group (lowercase letters) and the supplemented group (upper case letters) at p < 0.05. P week < 0.001; p genotype < 0.001; p vitamin D = 0.910; p week x genotype = 0.004; p genotype x vitamin D = 0.151; p week x vitamin D < 0.001; p week x genotype x vitamin D = 0.049.
The feed intake increased from the beginning of the trial in all genotypes and lastly reached a plateau in R11 hens at week 28 of life (Figure 4(a)). The feed intake of BLA hens increased markedly until week 24 of life (p < 0.05) and was higher compared to all other birds from week 22 until week 44 of life (p < 0.05). Thereafter, the feed intake of the different genotypes aligned except for the feed intake of R11 hens. A markedly increase of the feed intake in R11 hens was observed at first from week 20 to 22 of life (p < 0.05). From week 18 until week 26 of life these hens consumed less feed compared to the other genotypes. Thereafter, the feed intake became closer to the feed intake of L68 and WLA hens but remained significantly lower compared to BLA hens until the end of the trial (p < 0.05).
Considering the whole experimental period the pooled LSMeans of calcium intake of all genotypes fed 3000 IU vitamin D 3 were lower compared to the 300 IU vitamin D 3 supplemented hens (p < 0.05) (Figure 5(a)). The phosphorus intake was affected in an opposite manner (p < 0.05) except for WLA hens, which showed no significant difference between differently supplemented hens ( Figure 5(b)). In week 48, the phosphorus intake was higher in L68c hens compared to L68+ hens (p = 0.029). The daily calcium intake and the phosphorus retention tended to correlate with each other at week 49 (r = 0.242; p = 0.056). Furthermore, a negative correlation between the daily phosphorus intake and the phosphorus retention was detected (r = −0.364; p = 0.003).
Egg weight increased over time in all genotypes except for the egg mass of R11 hens, which seemed to decrease from week 22 to week 24 of life (Figure 4(b)). However, this effect was mainly based on the low number of laid eggs and was not significant (p > 0.05). Until week 40, the eggs of low performing hens were lighter compared to high performing hens (p < 0.05). Thereafter, the egg weight of L68 hens became closer to the egg weight of the high performing hens but the egg weight of R11 hens remained significantly lower compared to the high performing hens (p < 0.05). From week 54 of life, the egg weight of R11 hens was even lower compared to L68 hens (p < 0.05). From week 32 to week 50, the eggs of WLA hens were lighter than eggs of BLA hens (p < 0.05).
Initially, in all genotypes a marked increase of the daily egg mass was observed, however the high performing hens reached a plateau faster compared to low performing hens (Figure 4(c)). During the whole observation period, low performing animals showed a significantly lower daily egg mass compared to high performing hens (p < 0.05). Furthermore, L68 hens reached faster the plateau than R11 hens. In contrast to high performing hens the daily egg mass began to decrease from week 40 on resulting in a significantly lower daily egg mass in week 68 of life than determined in week 40 (p < 0.001). Considering the whole experimental period the daily egg mass of R11 hens was significantly increased by an additional vitamin D 3 supplementation (pooled LSMeans: R11c = 30.7 ± 0.4 g/d; R11+ = 33.5 ± 0.4 g/d; p < 0.001) and tended to be decreased in BLA hens (BLAc = 53.1 ± 0.4; BLA+ = 51.6 ± 0.04; p = 0.064). The feed conversion ratio decreased initially in all hens and became closer from week 28 on until the ratios began to differ once again between genotypes (Figure 4(d)). From week 42 on, the feed conversion ratio slightly increased in L68 hens, which in turn resulted in a higher ratio compared to the high performing hens (p < 0.05). In week 54 of life, also the R11 hens showed a higher feed conversion ratio compared to the high performing hens and at week 62 and 64 compared to WLA hens (p < 0.05). While the 3000 IU vitamin D 3 supplementation increased the feed conversion ratio in L68 hens at week 20 of life (L68c = 7.9 ± 0.6; L68+ = 17.1 ± 1.0 g/g; p < 0.001), in BLA hens the feed Figure 3. Laying period and egg production rate depending on vitamin D 3 supplementation in different genotypes (LSMeans ± SE, n = 36), (a) Laying maturity depending on genotype and vitamin D 3 supplementation (black−300 IU; white−3000 IU), (•, ○ -laying intensity of at least 50%); p genotype < 0.001; p vitamin D = 0.144; p genotype x vitamin D = 0.677 and end of laying period (▲, ∆ -laying intensity < 50% for at least three consecutive weeks after reaching the laying maturation); p genotype = 0.193; p vitamin D = 0.243; p genotype x vitamin D = 0.181. a-c; A-C Different superscripts mark significant differences between genotypes in the control group (lowercase letters) and the additionally supplemented group (upper case letters) at p < 0.05. (b) Duration of the laying period depending on genotype and vitamin D supplementation (black−300 IU; white−3000 IU); p genotype < 0.001; p vitamin D = 0.547; p genotype x vitamin D = 0.135. a-c; A-C Different superscripts mark significant differences between genotypes in the control group (lowercase letters) and the additionally supplemented group (upper case letters) at p < 0.05. (c) egg production rate depending on different genotypes and vitamin D supplementation (• -L68c (c, 300 IU); ■ -L68+ (+, 3000 IU); ○ -R11c; □ -R11+; ▲ -BLAc; ♦ -BLA+; ∆ -WLAc; ◊ -WLA+); p week < 0.001; p genotype < 0.001; p vitamin D = 0.454; p week x genotype < 0.001; p genotype x vitamin D = 0.068; p week x vitamin D = 0.045; p week x genotype x vitamin D = 0.026. conversion ratio was decreased by the higher vitamin D 3 supplementation (BLAc = 5.7 ± 3.9; BLA+ = 3.9 ± 0.2; p < 0.001). In week 24 of life, R11+ showed a higher feed conversion ratio compared to R11c hens (R11c = 5.0 ± 0.4; R11+ = 7.2 ± 0.3; p = 0.031). No further differences were observed between hens with and without additional vitamin D supplementation at specific time points but L68+ hens showed a significantly higher feed conversion ratio compared to L68c hens considering the whole experimental period (L68c = 3.2 ± 0.1; L68+ = 3.6 ± 0.1; p = 0.008).

Egg quality laying trial
Initially, the eggshell weight increased slightly. At each observed time point the eggshells were lighter in low performing hens compared to high performing hens irrespective of the vitamin D supplementation (p < 0.05) (Figure 6(a)). A significant week-by-genotype-by-vitamin D interaction was observed in case of eggshell thickness (p = 0.015) (Figure 6(b)). While the eggshell thickness in WLA hens was higher compared to the low performing hens from the beginning of the experiment (p < 0.05) except for WLA+ hens in week 52, the eggshell thickness of BLA hens increased at first from week 44 of life. The eggshell thickness of L68c remained on a low level during the whole experimental period. In contrast, the thickness in R11c and R11+ hens increased slightly from week 36 until week 52 (p < 0.05).
The breaking strength of eggshell decreased over the experimental period especially in WLA hens which initially showed significantly higher breaking strengths compared to the other hens (p < 0.05) and became closer to them over the experimental period ( Figure 6(c)). Pooled LSMeans of the breaking strength of eggs were higher in genotypes that received 3000 IU vitamin D 3 supplementation in week 36 of life (p = 0.020). Furthermore, considering all genotypes and the whole experimental period the pooled LSMeans of breaking strength of hens fed 3000 vitamin D 3 were higher compared to hens with lower vitamin D supplementation (p = 0.044).
The eggshell density increased from week 28 until week 44 in all hens (p < 0.001) (Figure 6(d)). At this period the eggshell density was higher in BLAc and BLA+ hens compared to the low performing hens (p < 0.05) except for the eggshell density in WLA+ hens in week 44. WLA+ hens showed higher eggshell density compared to low performing hens in week 60 (p < 0.05). Additionally, BLA+ hens showed higher eggshell density compared to R11+ hens in this week (p < 0.05).
There was a positive correlation between the breaking strength and the weight (r = 0.251; p < 0.001) as well as the thickness (r = 0.414; p < 0.001) of the eggshell.

Egg quality balance trial
During balance trial in week 29, there was no effect of oviposition time on eggshell thickness or eggshell breaking strength (p > 0.05) (Supplemental Table 2).
The proportions of eggshell, egg white and egg yolk differed between the genotypes (p < 0.05) but not between the two feeding groups but the higher vitamin  Table 3). The pooled LSMeans of eggshell proportion were significantly increased in R11 and WLA hens compared to BLA hens (p < 0.05). The pooled LSMeans of eggshell thickness were lower in R11 and BLA hens compared to L68 and WLA hens (p < 0.05). The pooled LSMeans of the breaking strength in WLA hens were higher compared to R11 and BLA hens and tended to be higher compared to L68 hens (p = 0.058). In week 29 of life, the breaking strength of eggshell tended to be increased by a vitamin D 3 supplementation of 3000 IU (p = 0.052).
A positive correlation between the eggshell proportion and the serum calcium (r = 0.365, p = 0.003) and phosphorus concentrations (r = 0.356, p = 0.004) were detected.
The pooled LSMeans of egg white proportion were significantly higher and LSMeans of egg yolk proportions were lower in BLA hens compared to the other genotypes (p < 0.001). The colour of egg yolk was more intensive in L68 hens compared to the other  genotypes (p < 0.05). No effect of the vitamin D supplementation on the yolk colour was observed (p > 0.05).

Discussion
Given the specific role of vitamin D for calcium homoeostasis of laying hens with egg(shell) output as the major calcium sink, it was hypothesised that the nutritionalphysiological requirement of 300 IU vitamin D 3 as recommended by GfE (1999) does not fit to the requirements of modern laying hybrids compared to hybrids with a genetically lower laying performance potential. The present study was performed to investigate whether an additional vitamin D 3 supplementation might have beneficial effects on the laying performance and egg quality of hens and therefore might support their genetic performing potential. BLA and WLA hens belonging to the high performing layer lines reached their laying maturity earlier compared to L68 and R11 hens belonging to the low performing layer lines. This aspect resulted in a longer duration of laying period. The laying period did not appear to be affected by the vitamin D 3 supplementation. However, it has to be emphasised, that only approximately 23% of all birds reached the actual end (laying intensity dropped below 50% per week for at least three consecutive weeks) of the laying period before the 70th week of life (end of the experimental trial). It cannot be ruled out that different completions and therefore different durations of the laying period between birds might become significant if all animals were observed until the actual end of the laying period.
Vitamin D 3 is involved in the transcellular and paracellular intestinal absorption of calcium (Adedokun and Adeola 2013). Adhikari et al. (2020) showed beneficial effects of high dietary vitamin D 3 concentrations on calcium digestibility. In the present study, no effect of the vitamin D 3 supplementation on the calcium retention was found; however, Adhikari et al. (2020) used concentrations which were double and three times higher than those used in the present study. The slight differences in calcium and phosphorus content between the laying-hen diets resulted in a lower calcium: phosphorus ratio and a lower calcium but a higher phosphorus intake over the whole laying period in hens consuming the diet containing 3000 IU vitamin D 3 . In studies of Kebreab et al. (2009) it was shown, that increased dietary calcium concentrations decreased the calcium retention in the body and egg, whereas phosphorus retentions increased. Especially in week 49, the lower calcium and higher phosphorus intake might be the explanation for the lower phosphorus retention in hens consuming the 3000 IU vitamin D 3 diet. This assumption was supported by the correlations between the intake of both major elements and the phosphorus retention in this week. Rodehutscord et al. (2002) traced a reduced phosphorus utilisation and net absorption in consequence of a higher calcium supplementation to a reduced phytate hydrolysis. Adedokun and Adeola (2013) explained that high dietary calcium increased the pH of the gastrointestinal content, which adversely affects the phytase efficacy. Consequently, the effects of the vitamin D 3 supplementation on phosphorus retention might have been influenced by the diet formulation and were not only direct vitamin D 3 effects, because vitamin D 3 is normally to a high extent involved in the activation of genes, which are responsible for the phosphorus transport in the intestine (Hattenhauer et al. 1999;Adedokun and Adeola 2013). It cannot be fully explained why this effect was not detected in week 29 of the present trial; however it is assumed that this might be based on the changes in the laying performance. Nevertheless, a lower phosphorus retention contributes to a higher environmental pollution which might also be considered with regard to the selection of the layer line. Especially at the beginning of the laying period in week 29, a higher phosphorus retention was shown in WLA hens compared to BLA hens which might be based on the differences in the calcium and phosphorus metabolism between the layer lines. Also, Adedokun and Adeola (2013) emphasised the influences of age, sex, productions and genetics on the metabolism of these minerals. A compensatory effect of vitamin D supplementation on these impact factors cannot be supported by the present results.
In the present study, it was clearly demonstrated that a higher vitamin D 3 supplementation leads to the 25-OHD 3 enrichment in serum of all used layer lines. Already Soares et al. (1995) reported correlations between the dietary vitamin D intake and the 25-OHD 3 concentrations in blood. In week 49, especially in white layer lines the 25-OHD 3 enrichment in serum was smaller than in the other analysed weeks. The 25-OHD 3 is the major vitamin D 3 form in the circulation (Soares et al. 1995;de Matos 2008). A decrease of this form might indicate that vitamin D 3 is converted to a higher extent to the most metabolically active form 1, 25-dihydroxy vitamin D 3 or to 24, 25-dihydroxy vitamin D 3. Both were; however, not measured in the present study. An up-regulation of the 1, 25-dihydroxy vitamin D 3 would be supported for example in case of hypocalcaemia but in case of high calcium and phosphorus concentrations, the major hydroxylation product is 24, 25-hydroxy vitamin D 3 (de Matos 2008; Proszkowiec-Weglarz and Angel 2013). Indeed it might be possible that lower calcium intake of hens fed 3000 IU vitamin D 3 provoked hypocalcaemia. Hypocalcaemia is also associated with an increased urinary phosphorus excretion, which is important for the maintenance of the blood phosphorus balance (Soares et al. 1995). Therefore, hypocalcaemia might also be a further explanation for the lower phosphorus retention in white layer lines, but not in brown layer lines which showed no reduced enrichment of 25-OHD 3 serum concentrations after the 3000 IU vitamin D 3 supplementation. The correlations between the phosphorus retention and the blood 25-OHD 3 concentrations in week 49 supported possible interrelations between both parameters. Presented calcium plasma levels were higher compared to reference values (3.3-5.9 mmol/l ≈ 13.2-23.6 mg/dl) for domestic fowls (Gallus gallus) described by Moritz (2014). Therefore, there were no indications of hypocalcaemia. Nevertheless, it is possible that hens suffered from calcium deficiency. As summarised by de Matos (2008) a high or low calcium: phosphorus ratio increases the requirements for vitamin D 3 in birds. According to the present results of the feed analyses the calcium: phosphorus ratio was lower in the 3000 IU vitamin D 3 group compared to the group with lower vitamin D 3 content. This might also have evoked a higher vitamin D 3 requirement in these birds especially in the middle of the laying period being characterised by a high laying performance and as indicated by a high eggshell density and weight also by a high eggshell mineralisation.
Hypovitaminosis of vitamin D 3 can also provoke signs of a calcium deficiency in birds (de Matos 2008). In the present trial; however, there were no adverse effects on calcium retention in week 49, which would have been expected in this case. Nevertheless, the blood 25-OHD 3 concentrations of control groups of the present study were below reference ranges of domestic chicken reported by de Matos (2008). Especially, in week 29 the vitamin D values of all control hens were extremely low while the calcium levels were increased. Elevated activity of ALP might indicate a calcium deficiency in this period especially in WLAc and BLAc hens. de Matos (2008) described that in case of calcium deficiencies, generally ALP is elevated and calcium concentrations are normal or elevated based on the higher activity of the parathyroid hormone. In week 29, the vitamin D 3 supplementation seemed to decrease the ALP activity in the high performing layer lines although the difference was not significant in WLA hens. In studies of Attia et al. (2020) no effect of vitamin D 3 supplementation on ALP values in serum were observed in brown egg layers which were supplemented with different calcium levels. However, these authors chosen lower values of vitamin D 3 concentrations and birds were older than birds used in the present study. ALP activity of 60-72-week old hens of Attia et al. (2020) equalled ALP activity of 69-old brown layers from the present study but were markedly lower than ALP activity of 29-old high performing hens receiving 300 IU vitamin D 3 . As a consequence, this could mean that especially in high performing layer lines an additional vitamin D supplementation might support the calcium metabolism in an early stage of the laying period. This might be also an explanation for the decrease of the feed conversion ratio in this period in BLA+ hens. The temporary increase of the feed conversion ratio of L68+ and R11+ hens is mainly based on the fact, that only a low number of hens reached the laying maturity at this time point and therefore showed no constant oviposition as well as a low and volatile daily egg mass production at this period. The fact that considering the whole experimental period L68+ hens exhibited a higher feed conversion ratio compared to L68c hens attracted more attention. A concrete explanation is not possible presently. However, the nutrient retention of L68 hens was lower compared to the other genotypes especially in week 49 which might support the effects on the feed conversion ratio.
Considering the whole experimental period R11+ hens exhibited a higher daily egg mass production compared to R11c hens despite similar individual egg weight. This indicates an improved constant production or higher number of produced eggs laid from R11+ compared to R11c hens, which was also shown by the constant egg production rate also in the later laying phase. Atencio et al. (2006) showed also a stabilisation of the daily egg production by a higher vitamin D 3 supplementation in broiler breeders especially at a later stage of the laying period. The decrease of the daily egg mass production in BLA+ compared to BLAc might be based on the numerically lower egg weight. It is suggested, that because of their higher calcium and vitamin D requirements the deficiency of calcium in the 3000 IU vitamin D 3 diet lead to the lower performance of BLA+ hens. In contrast, R11 hens are in general low performing hens, so that the lower calcium intake might be balanced by the higher vitamin D 3 supplementation in R11+ hens.
The enrichment of 25-OHD 3 in serum was also reflected in the egg yolk. Both the total vitamin D 3 and 25-OHD 3 increased with 3000 IU vitamin D 3 supplementation. The amount of the enrichment in egg yolk was similar to the vitamin D enrichment in indoor housed hens which were exposed at least 3 h with artificial ultraviolet B light (Kühn et al. 2019). BLA+ hens reached also amounts of total vitamin D 3 and 25-OHD 3 in egg yolk which equalled the amounts in egg yolk of hens which could choose freely between inside and outside pens (Kühn et al. 2014). As described by de Matos (2008) also in the present study vitamin D 3 showed a higher affinity to be deposited into the egg yolk than 25-OHD 3 . Although the difference was not significant the numerically higher serum 25-OHD 3 in BLA+ hens might be the explanation for the higher vitamin D 3 and 25-OHD 3 concentrations in egg yolk of these hens. Unfortunately, only in week 29 the vitamin D concentrations in egg yolk were determined. It would have been interesting to know, if the lower serum 25-OHD 3 concentrations were also reflected in the egg yolk in week 49. The higher deposition of vitamin D 3 with increasing dietary vitamin D 3 levels was also shown in studies of Yao et al. (2013).
From an economic point of view, the eggshell quality including eggshell weight, percentage, thickness, strength and density is extremely important because of its crucial role in egg production and hatchability (Ketta and Tumová 2016). Internal factors affecting eggshell quality described in the literature (Ketta and Tumová 2016) such as age and genotype were also in the present study prominent impact factors on eggshell quality. An increased eggshell weight is associated with increased egg size related to an increased shell surface area (Ketta and Tumová 2016). These relationships are also the explanation for the differences in the egg shellweight between high and low performing hens. Especially with regard to the eggshell strength WLA hens seemed to be superior compared to the other genotypes at the beginning of the observations which seemed to be mainly related to the shell thickness indicated by the positive correlation between both parameters. This relationship was already demonstrated in studies of Tumova et al. (2014). At no time point the other genotypes reached the breaking strength of WLA eggs measured in week 28 or 36. Corresponding to Pavlík et al. (2009) eggshell breaking strength decreased in an age dependent manner. They discussed relationships between the blood calcium levels and the eggshell breaking strength as well as the eggshell thickness but found no relations on blood phosphorus levels (Pavlík et al. 2009). In the present study a positive correlation between the eggshell proportion and serum calcium and phosphorus concentrations was detected but no correlation between eggshell strength and calcium or phosphorus concentrations in serum in week 29 (data not shown). Considering all genotypes and the whole experimental period the pooled LSMeans of breaking strength of hens fed 3000 IU vitamin D 3 were higher compared to hens with lower vitamin D supplementation. Although they did not concretely determine the breaking strength of the eggshell, also Atencio et al. (2006) reported a softer eggshell of eggs of broiler breeders receiving low amounts of vitamin D 3 .

Conclusion
The higher vitamin D 3 concentrations in feed used in the present study might have beneficial effects on the calcium and phosphorus homoeostasis in the early laying period of high performing layer lines. In turn, the feed efficiency can be improved. Additionally, the higher vitamin D 3 supplementation seemed to lead to an improved constant production of eggs in R11 hens. In general, positive effects appeared to be influenced to a high extend by the genotype and laying performance of birds. However, further studies need to guarantee a balanced calcium-phosphorus content in the feed. The increase of serum 25-OHD 3 by the higher vitamin D 3 supplementation supported the higher transfer of vitamin D in the egg yolk and improved genotype-dependently the breaking strength of the eggshell. Therefore, the present study gives evidence that there are economic advantages and nutritional-physiological benefits for hens and consumers of eggs with a higher vitamin D 3 supplementation in laying hens especially at the beginning of the laying period.