The trial was approved by the ethics commission of the Austrian Ministry of Science and Research (BMWF-66.016/0010―WF/V/3b/2016) and of the Austrian Agency for Health. In total, 24 Lohman Brown-Classic laying hens (35 weeks old) with an initial body weight of 2000 ± 269 g purchased from a commercial breeder were subjected to this experiment. To minimize the influence of body weight, the individuals were weighed, leg-tagged and distributed to six feeding regimes according to their body weight, randomly. Each hen was accommodated in one metabolism cage equipped with a wooden perch, a drinker and an attached laying nest. The cages enabled controlled feeding and birds were exposed to light for 16 h per d. Treatments were arranged as a 3 × 2 factorial design, with three WB concentrations (0 g∙kg⁻¹, 75 g∙kg⁻¹, and 150 g∙kg⁻¹) and two kinds of vegetable oil (rapeseed oil and sunflower oil). The feed intake was limited to 110 g per d. The hens were fed equal amounts twice daily (8:00 AM and 5:00 PM). Water was provided ad libitum for all treatments and the individual health status of all laying hens was recorded daily. Each of the six treatments was studied with four laying hens. The diets were calculated to reach balanced metabolizable energy (11.4 AME_N MJ ME/kg), crude protein (170 g∙kg⁻¹), and digestible amino acids content (Table 1). Nutrient concentration was calculated to meet or exceed the nutritive requirements for laying hens of the manufacturer (Lohmann, Cuxhaven, Germany).

¹CON R = 0 g∙kg⁻¹ wheat bran, rapeseed oil; CON S = 0 g∙kg⁻¹ wheat bran, sunflower oil; LOW R = 75 g∙kg⁻¹ wheat bran, rapeseed oil; LOW S = 75 g∙kg⁻¹ wheat bran, sunflower oil; HIGH R = 150 g∙kg⁻¹ wheat bran, rapeseed oil; HIGH S = 150 g∙kg⁻¹ wheat bran, sunflower oil; ²Vitamins per kg premix: 4 × 10⁷ IU vitamin A, 1.7 × 10⁷ IU vitamin D3, 1.7 × 10⁵ mg vitamin E, 1.4 × 10⁴ mg vitamin K, 1 × 10⁵ mg vitamin B1, 2.5 × 10⁴ mg vitamin B2, 1.5 × 10⁴ mg vitamin B6, 75 mg vitamin B12, 2.3 × 10⁵ mg nicotinic acid, 65 mg pantothenic acid, 6.500 mg folic acid and 400 mg biotin. ³Trace elements per kg premix: 120 g Fe, 120 g Zn, 180 g Mn, 30 g Cu, 2 g I, 2 g Co and 0.8 g Se. ⁴ZY-Phytase®; ⁵Rovabio®.

The experiment lasted for 21 ds. After an adaption phase of 15 ds, eggs were individually collected from each animal, weighed and subjected to further analyses.

Collected eggs (n = 144) were inspected for visible dirt (0 = no dirt, 1 = moderate dirt, 2 = severe dirt), and monitored regarding egg weight and shell strength using a texture analyzer (TA.HD.plus Texture analyzer, Hamilton, USA). Afterwards, eggs were broken and yolk and albumen weight determined. Yolk color (L*, a*, b*) was measured using a reflectance colorimetry spectro-photometer (CM-600d; Konica Minolta, Tokyo). Haugh units were calculated according to the formula HU = 100 × log (H + 7.57 − 1.7W^0.37). The height of the albumen was denoted as H, while W was the weight of the egg [11] . Shell weight and thickness (including shell membrane) were registered after rinsing under tap running water and drying overnight at 103˚C. Shell thickness was determined on two randomized points on the poles of the eggshell. Subsequently, three yolks of the same animal were pooled (collected at ds 16 - 18 and 19 - 21), respectively. Pooled and homogenized yolks were freeze-dried overnight using an Edwards Modulyo 4K freeze drier (Edwards, Kniese and Co., Marburg, Germany). Dry matter was determined by back-weighing the lyophilisate. The dried yolk was manually powdered in a stone mortar and stored at −80˚C for further analyses. Representative samples of diets, taken at the beginning of the feeding trial from all tested diets, were ground in a Retsch ultra-centrifugal mill of type ZM1 (Haan, Germany) with a 1 mm sieve. Regarding the dry matter, ash, crude protein (CP) and ether extract (EE) all samples of eggs and feed were analyzed in duplicates according to the official methods of VDLUFA [12] . Additionally, samples of feed underwent starch, sugar and neutral detergent fiber (NDF) analyses according to Naumann and Bassler [12] . Phytic acid was determined according to Makkar et al. [13] , and gross energy in a bomb calorimeter under oxygen conditions (IKA-Kalorimeter C400, Bartelt, Graz, Austria). Calcium, sodium, and zinc were analyzed by means of atomic absorption spectrophotometry (AAnalyst 200, Perkin Elmer, Brunn am Gebirge, Austria). After the previous wet-ashing in a microwave oven (MLS-ETHOS plus Terminal 320, Leutkirch, Germany), phosphorus was determined using the vanado-molybdate method [12] based on absorption measurement at 436 nm (U5100-Spectrophotometer-Hitatchi, Metrohm, Wien, Austria).

Pooled yolk samples and corresponding feed samples were analyzed according to the method of Schreiner [14] . In brief, fatty acid methyl esters (FAME) were obtained from freeze-dried samples by a combined extraction/tramsmethylation. An amount of 50 mg yolk powder was weighed into a 15 ml reaction tube and 2 mL of toluene containing 1 mg/ml of each internal standard (C17 methyl ester and alpha -cholestane) was added. For transmethylation, 3 ml of methanolic HCl, 5% (v/v) were supplemented and samples were placed in a 70˚C water bath for two hours with occasional shaking by hand. After transmethylation, 5 mL of 6% calcium carbonate were added and the mixture was centrifuged gently for 5 mins in order to obtain phase separation. The upper phase was transferred to another reaction tube where the solvent was removed by a stream of N2. To the dry sample, 100 µl BSTFA/TMCS and 100 µl pyridine were added for masking the hydroxyl group of cholesterol. Derivatization was obtained at 60˚C in a drying oven for 30 mins. After reaction, the samples were dissolved in 2 mL toluene. Gas chromatography was performed on a Thermo scientific Trace GC Ultra system (Waltham, USA) using a RTX-225 capillary column (30 m lengh; 0.25 mm ID). Restekcorp 800-356-1688 Hydrogen was used as carrier gas at a flow rate of 3 ml/min and a head pressure of 90 kPa. Detection was performed via an autosampler and the temperature program was 120˚C for one min followed by a 20˚C/min ramp to 170 and continuing with a 2.5˚C/min ramp to 220. This final temperature was constant for one min. Fatty acid methyl ester were determined by comparison with appropriate standards and no response factors were used for calculation.

Alpha-Tocopherol analyses in pooled yolk and diet samples were performed according to Panfili et al. [15] with minor modifications. For saponification, 500 mg of freeze-dried yolk powder or 1000 mg of feed samples were placed in a reaction vial and 2 ml of potassium hydroxide (600 g/l), 2 ml ethanol (95%), 2 ml of sodium chloride (10 g/l), and 5 ml of ethanolic pyrogallol (60 g/l) added. Subsequently, after 45 min of saponification at 70˚C, tubes were cooled in an ice bath, 15 ml of sodium chloride (10 g/l) was added and followed by extraction with 15 ml of n-hexane/ethyl acetate (9:1 v/v). This procedure was conducted twice and the organic layer was recollected and evaporated to dryness. To dissolve the residue, 2 ml of n-hexane were used. Chromatography was performed using an HPLC analytical system (Shimadzu, Kyoto, Japan) equipped with a fluorescence detector (295 nm EM and 354 nm EX). An amino phase column was used for separation with a mobile phase composed of 70% n-hexane and 30% ethylacetate.

Intact eggs were aseptically transferred into sterile stomacher bags and treated as described by Musgrove et al. [16] . In detail, one egg was placed per bag and 30 ml buffered peptone water added. After 5 min of incubation, the egg was manually rubbed for one min to release the microorganisms adhering to the surface into the liquid phase. After the removal of the egg from the bag under sterile conditions, the solution was mixed thoroughly by stomaching the content for 60 sec. Then 250 µl of Salmonella supplement (bioMérieux Ref. 42650) were added and the bag was incubated for 20 h at 41.5˚C in order to selectively enrich the microorganisms.

Following these enrichment procedures, the VIDAS^TM UP Salmonella (SPT) Assay (bioMérieux, Marcy l’Etoile France) was performed, throughout following the manufacturer’s instructions. Briefly, 500 µl of the enriched solution was applied to the single-dose reagent strip, and each strip was heated at 131˚C for 5 min using a VIDAS^TM Heat & Go dry heating system (bioMérieux, Marcy l’Etoile, France). After cooling to room temperature for 10 min, strips were inserted into the mini VIDAS^TM instrument (bioMérieux, Marcy l’Etoile, France), and the detection procedure was initiated. Results were automatically calculated and denoted as negative or positive per 3 g per eggshell surface.

Data were evaluated with a two-way ANOVA using the MIXED procedure of SAS Enterprise Guide 7.1, according to the following model: xijk = µ + αi + βi + (αβ)ij + Ak + eijk, x = dependent variable, µ = overall mean, α = effect of WB (0 g∙kg⁻¹, 750 g∙kg⁻¹ and 150 g∙kg⁻¹), β = effect of oil (rapeseed oil or sunflower oil), Ak = random effect of animals (k = 1, 2, ∙∙∙, 24), eijk = residual experimental error. Multiple comparisons of the means were determined using the Tukey Kramer test. Treatment means were presented as least-squares means, and significant differences in the means are displayed with different superscripts. The influence of the diets on the categorical parameter “dirtiness of eggshells” (D) treated as ordered multinomial response was calculated according to the GLIMMIX procedure (considering the random effect of the individuals) using SAS Enterprise Guide 7.1.

A detailed description of the chemical composition and the fatty acid profile of the six diets applied in this study are presented in Table 2 and Table 3. Aiming at a theoretically calculated protein concentration of 170 g/kg, the analyzed crude protein levels were somewhat higher but almost similar, practically ranging between 177 g/kg and 182 g/kg. Upon calculation, the target AME_N value of 11.4 was slightly lower compared to the analyzed values of the diets containing no bran (CON R: 11.7 MJ/kg; CON S: 11.9 MJ/kg) and 75 g/kg bran (LOW R: 11.6 MJ/kg; LOW S: 11.7 MJ/kg). Due to lower starch and higher neutral detergent fiber (NDF) levels in those diets containing higher amounts of bran, it was necessary to add more oil to meet the energy requirements. Consequently, ether extract (EE) levels increased in parallel with the amount of WB. Correspondingly, higher phytate contents were found owing to higher amounts of WB in the diet, i.e. CON R showed a phytate content of 10.8 g/kg, whereas HIGH R displayed a level of 11.6 g/kg. As a logical consequence of the oil supplementation, the amounts of saturated fatty acids (SFA), polyunsaturated fatty acids (PUFA), omega-6 polyunsaturated acids (∑n−6 PUFA) and the percentage distribution between ∑n−3 PUFA and ∑n−6 PUFA (∑n−6 PUFA/∑n−3 PUFA) were higher in

¹CON R = 0 g∙kg⁻¹ wheat bran, rapeseed oil; CON S = 0 g∙kg⁻¹ wheat bran, sunflower oil; LOW R = 75 g∙kg⁻¹ wheat bran, rapeseed oil; LOW S = 75 g∙kg⁻¹ wheat bran, sunflower oil; HIGH R = 150 g∙kg⁻¹ wheat bran, rapeseed oil; HIGH S = 150 g∙kg⁻¹ wheat bran, sunflower oil; ²AME_N = metabolizable energy

¹CON R = 0 g∙kg⁻¹ wheat bran, rapeseed oil; CON S = 0 g∙kg⁻¹ wheat bran, sunflower oil; LOW R = 75 g∙kg⁻¹ wheat bran, rapeseed oil; LOW S = 75 g∙kg⁻¹ wheat bran, sunflower oil; HIGH R = 150 g∙kg⁻¹ wheat bran, rapeseed oil; HIGH S = 150 g∙kg⁻¹ wheat bran, sunflower oil; ²SFA= saturated fatty acids; MUFA = mono unsaturated fatty acids; PUFA = polyunsaturated fatty acids; ND = not detectable; TUFA = total unsaturated fatty acids.

the diets containing sunflower oil, whereas monounsaturated fatty acids (MUFA), and omega-3 polyunsaturated acids (∑n−3 PUFA) showed lower concentrations. Regarding the α-tocopherol, phosphorus and calcium concentrations in the diets, slight differences obviously were caused by analytical variability resulting from unavoidable small sample weights.

As presented in Table 4 no differences were observed across the treatments regarding feed conversion ratio, live weight changes and egg mass. These results are in accordance with those of Ruan et al. [17] . Furthermore, no specific feed refusal due to the wheat bran containing diets was observed during the experiment, as daily feed intakes did not differ.

External and internal egg quality parameters are presented in Table 5. There were no differences across the different supplementations regarding egg weight, shell weight, albumen weight, yolk weight, haugh unit, shell thickness and eggshell breaking strength, regarding the dirtiness of the eggshells.

Diets comprising high amounts of fiber usually might increase fecal viscosity and cause watery and sticky excrements [18] . According to the present findings, neither the high nor the low WB level diet negatively influenced the cleanliness of the eggshells. On the contrary, in comparison with the control treatment, WB implementation rather led to cleaner eggshells. In fact, the control group showed 2.6 times more dirty eggshells compared to the HIGH treatment, while the LOW treatment displayed a factor of 1.6, respectively (data not shown). Hence, we assume that 150 g∙kg⁻¹ WB in the diet did not alter the fecal viscosity and can be regarded as an appropriate proportion ensuring satisfactory egg surface quality, if NSP-hydrolyzing enzymes are included in the diet. This finding is of relevance in terms of consumer acceptance and marketability of the eggs.

¹CON: 0 g∙kg⁻¹ wheat bran; LOW: 75 g∙kg⁻¹ wheat bran; HIGH: 150 g∙kg⁻¹ wheat bran; RSO: rapeseed oil; SFO: sunflower oil; ²= NS: p-value > 0.1; * = p-value < 0.1; ** = p-value < 0.05; Identical superscripts represent no significant difference whereas differing letters indicate a significant distinction. n = 24 (6 × 4); Egg mass: n = 144 (6 × 4 × 6).

¹CON: 0 g∙kg⁻¹ wheat bran; LOW: 75 g∙kg⁻¹ wheat bran; HIGH: 150 g∙kg⁻¹ wheat bran; RSO: rapeseed oil; SFO: sunflower oil; ²NS = p-value > 0.1; *= p-value < 0.1; **= p-value < 0.05; Identical superscripts represent no significant difference whereas differing letters indicate a significant distinction. Egg: n = 144 (6 × 4 × 6); Yolk: n = 48 (6 × 4 × 2).

Across the different diets, no differences were observed regarding pH-value, dry matter, protein and fat contents as well as lightness L*. As presented in Table 5, increasing WB contents in the diet correlated with decreased yolk redness and yolk yellowness.

The diverse egg quality parameters examined in this study suggest that both a 75 g∙kg⁻¹ and a 150 g∙kg⁻¹ of WB proportion in the diet are appropriate, as no adverse effects on egg quality were observed. These findings are in agreement with those of Ruan et al. [18] , who did not report any negative impact of diets containing 180 g∙kg⁻¹ rice bran on duck egg quality and yolk color. Likewise, Incharoen and Maneechote [19] found that 30 g∙kg⁻¹ and 60 g∙kg⁻¹ of whole rice hulls resulted in acceptable egg quality, but in decreased yellowness of the yolk. It is well known that corn provides high amounts of xanthophylls, which are responsible for the yellow color of the yolk. Hence, theoretically and also supported by the present data, corn substitution by WB usually results in a reduced level of xanthophylls and thus in lower color intensity.

The two oil sources used did not influence the yolk color. This finding is in contrast to that of Ceylan et al. [5] , who observed a decrease in yolk color intensity, when sunflower oil was applied in comparison to rapeseed oil. Dalle Zotte et al. [20] claimed that yolk color can be modulated by the addition of various oils, mainly based on the natural pigments (e.g., oxicarotenoid in linseed oil). In this context, it should be stressed that different methods for measuring the yolk color may yield different results. For example, Ceylan et al. [5] used a Roche Yolk Color Fan for determining the yolk color, while in our study yolk color was assessed by means of reflectance colorimetry. Having some impact on the marketability of the eggs, we conclude that neither WB supplementation nor the two different oils used exert negative effects on yolk color.

No Salmonella contaminations were detected in the eggs as well as in the faeces throughout the experiments. We assume that this was due to the good hygiene quality of the feed. Moreover, there is some evidence, that a diet high in fiber may promote some enhanced resilience against Salmonella infections [28] .