Effect of fibrous diet and vitamin C inclusion on uniformity, carcass traits, skeletal strength, and behavior of broiler breeder pullets X. Asensio,*,y,1 N. Abdelli,* J. Piedrafita,z M. D. Soler,x and A. C. Barroeta* *Animal Nutrition and Welfare Service (SNIBA), Department of Animal and Food Science, Faculty of Veterinary onoma de Barcelona 08193, Bellaterra, Barcelona, Spain; yAviagen S.A.U. 08184 Pal- Medicine, Universitat Aut a i Plegamans, Barcelona, Spain; zDepartment of Animal and Food Science, Faculty of Veterinary Medicine, au-solit Universitat Autonoma de Barcelona 08193, Bellaterra, Barcelona, Spain; and xDepartment of Animal Production and Health, Public Veterinary Health and Food Science and Technology, Faculty of Veterinary Medicine, Universidad CEU Cardenal Herrera, CEU Universities, Valencia, Spain ABSTRACT This experiment studied the effect of ash content values (P , 0.05). They also had lower ALP broiler breeder nutritional strategies on uniformity, carcass serum level at 6 and 22 wk (P , 0.05), their breast muscle traits, tibia parameters, and behavior during rearing and was less developed (18.5 vs. 19.8%, P , 0.05), and their prebreeder periods (up to 22 wk of age). One-day-old pul- abdominal fat deposition was higher (1.14 vs. 0.87%, lets (n 5 384) were randomly assigned to 4 treatments P , 0.05). At 15 and 22 wk, they performed, on average, arranged as a 2 ! 2 factorial, with 2 fiber levels (control vs. 97% less grasping feather pecking and 45% less non–food fibrous diet, 15% diluted in AMEn and nutrient content) object pecking behaviors, and their wing-feather score and 2 vitamin C feed inclusions (0 vs. 200 mg/kg). At 6, 15, was lower (P , 0.05) at 22 wk. Tail- and wing-feather and 22 wk, blood sampling was carried out (4 birds/repli- scores of the control treatments were reduced by vitamin cate) to determine serum alkaline phosphatase (ALP) C inclusion (tail: 0.30 vs. 1.15, P , 0.05; wing: 0.98 vs. 1.26, levels, and behavior was observed by visual scan sampling. P , 0.05) at 22 wk. In conclusion, fibrous diet improves At 22 wk, carcass traits, tibia parameters, and intestinal carcass traits (reduces breast muscle and increases morphology were assessed (2 birds/replicate), and tail- and abdominal fat deposition), deteriorates bone mineral wing-feather integrity of all birds were scored. Fibrous diet deposition and thus skeletal strength, and reduces stereo- did not modify BW uniformity, mortality, or tibia growth typic behaviors, improving wing-feather integrity. Vitamin when compared with control diet. Pullets fed the fibrous C inclusion improves tail- and wing-feather integrity of diet had lower tibia breaking strength, elastic modulus, and lower in feed allowance. Key words: broiler breeder, fibrous diet, vitamin C, skeletal strength and behavior 2020 Poultry Science 99:2633–2644 https://doi.org/10.1016/j.psj.2020.01.015 INTRODUCTION advised standard BW and not having received the nutri- ents required, thus leading to poor body development and Correct management of broiler breeders is essential to stressful situations, which, respectively, might cause leg prevent having uneven flocks, where larger or aggressive health and stereotypic behavior issues. pullets are likely to outcompete smaller or timid pullets, Fibrous (FBR) diets used to dilute the daily distrib- resulting in unequal access to feed and increasing flock uted ration, by adding different concentrations of raw BW variation (Zuidhof et al., 2015). Low uniformity material sources of insoluble fiber, might be a useful nutri- may result in broiler breeder pullets being under the tional strategy to increase daily feed allocation and cleanup time. In this way, it might be possible to obtain more uniform flocks and reduce pullets under the stan- Ó 2020 The Authors. Published by Elsevier Inc. on behalf of Poultry dard BW with poor skeletal development and under Science Association Inc. This is an open access article under the CC BY- stress. Likewise, Gonzalez-Alvarado et al. (2007) found NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). that the gizzard contents were greater in broilers chicks Received September 13, 2019. Accepted January 26, 2020. fed oat and soy hulls, sources of insoluble fiber, than in 1 Corresponding author: xasensio@aviagen.com chicks fed the control (CTR) diets, indicating that fiber 2633 2634 ASENSIO ET AL. was retained longer in this organ; Hetland and Svihus The aim of the present study is to assess the effect of (2001) also found that inclusion of 4% oat hulls increased FBR diet (15% diluted in AMEn and nutrient content) the fresh contents of the gizzard in chicks. In broiler and vitamin C supplementation (200 mg/kg) on BW uni- breeders, longer filling of the gizzard, in particular, and formity, mortality, carcass traits, tibia parameters, and of the gastrointestinal tract, in general, might promote behavior of broiler breeders in the rearing and prebreeder a feeling of satiety and reduce stress mainly during the phases. We hypothesized that there would be greater period of rearing when weekly feed increases are lower. skeletal strength and lower stereotypic behaviors than Vitamin C, which is not normally included in broiler those in control pullets. Furthermore, ALP and tail- breeder premixes, has to be studied as another nutritional and wind-feather scores have been proposed to assess strategy. Regarding body development, when vitamin C is skeletal development and stress, respectively, in broiler supplemented, it enhances 1,25-dihydroxyvitamin D breeder pullets. (1,25-D) production (Weiser et al., 1988), which influences calcium metabolism and thus affects bone growth. In addi- MATERIALS AND METHODS tion, its deficiency results in widespread connective tissue abnormalities, whose origin comes from a disruption in Birds and Facility collagen synthesis (Whitehead and Keller, 2003). Vitamin C supplementation might also be useful because, although All of the animal experimentation procedures used in poultry can biosynthesize it, its endogenous synthesis may the experiment were approved by the Animal Ethics not be adequate to always meet the full poultry require- Committee of the Universitat Autonoma de Barcelona ments, specifically during stress periods, when they may (UAB, Barcelona, Spain) and were in compliance with exceed the synthesizing ability (Gous and Morris, 2005). the European Union guidelines for the care and use of an- Furthermore, vitamin C may also help the broiler breeders imals in research (European Parliament, 2010). to cope with stressful situations (Satterlee et al., 1993, A total of 384 one-day-old Ross 308 female broiler 1994; Jones et al., 1996, 1999), mainly when breeder chicks were randomly distributed into 32 pens management is suboptimal during the rearing period. (12 chicks/pen) in the experimental facility Granja To evaluate whether skeletal development of the broiler Sole (Vila-rodona, Tarragona, Spain) during a period breeders during the rearing phase is correct, pullet BW has from 0 to 22 wk of age. Each pen had an area of 1.5 m2 to be weekly compared with the BW profiles provided by and contained a bell drinker. Feed and drinker space the breeding companies; however, this is an indirect form was sufficient for all the hens to reach feed and water to assess correct skeletal growth. Alkaline phosphatase at the same time. New wood shavings were utilized as (ALP) is a serological marker directly related to skeletal litter material, and litter was topped up weekly to keep development and, in particular, to bone formation and it dry. growth (Seibel, 2005). ALP is a ubiquitous enzyme A vaccination program adapted to the epidemiology attached on the outer cell surface (Stinson and of the farm area was followed throughout the rearing Hamilton, 1994); the precise function of the enzyme is still period. Management applied was in accordance with unknown, but it obviously plays an important role in the guidelines of the Ross Parent Stock Management osteoid formation and mineralization (Harris, 1990). Handbook (Aviagen, 2013). This serological marker is now an important tool in the assessment and differential diagnosis of metabolic bone Experimental Design diseases in humans; nevertheless, it has never been used in broiler breeder pullets to assess body development dur- The experiment included 4 treatments arranged as a 2 ing the rearing period. ! 2 factorial with 2 fiber levels, CTR diet andFBR diet, Physiological indicators of stress, such as plasma corti- and 2 vitamin C inclusions, diet not vitamin C– costerone concentrations, heterophil-to-lymphocyte ratio, supplemented (C2) and diet vitamin C–supplemented and white-blood-cell frequencies, have been used to eval- (200 mg/kg) (C1). Therefore, the treatments were uate the welfare of broiler breeder pullets (Savory et al., CTR/C2, CTR/C1, FBR/C2, and FBR/C1, and 1996; Hocking et al., 1998, 1999; de Jong et al., 2002; each one was replicated 8 times. Sandilands et al., 2006). Apart from these indicators, a Birds were randomly allotted to the different repli- noninvasive measurement would be practical as a test to cates, with an initial BW of 43.2 g 6 2.94 g and a BW quickly assess the ease or uneasiness of a flock, for coefficient of variation (CV) of 7.1 6 0.51%. example, stress of the broiler breeder pullets in the rearing farms. Morrissey et al. (2014) used feather pecking Feeding Program, Diets, and Feed Intake as a parameter to determine stress and welfare, and their study showed that broiler breeders fed FBR diluted diets Feed was produced in the feed mill of Institut de feather-pecked significantly less often. Therefore, because Recerca i Tecnologia Agroalimentaria Mas Bove (Con- feather pecking results in damaged feather cover, the stantí, Tarragona, Spain). A four-phase diet-feeding pro- assessment of tail- and wing-feather integrity might be a gram was followed: Starter I (the first week), Starter II practical way to evaluate stress and, if necessary, to (from 2 to 6 wk), Grower (from 7 to 15 wk) and Pre- quickly apply corrective management actions. breeder (from 16 to 22 wk). Starter I was the only diet FIBROUS DIET AND VITAMIN C IN BREEDERS 2635 shared by the 4 treatments. Feed-form presentation was calculated by multiplying all of the pullets present at crumble for Starter I, short pellet for Starter II, and reg- that moment in the pen by the daily AMEn allowance ular pellet for Grower and Prebreeder. Feed was always per bird and divided by the AMEn of the diet. Both, stan- distributed on the floor. dard BW profile and daily AMEn allowance per pullet, Ingredients and nutrient composition of the experi- were based on Ross 308 Parent Stock Performance Objec- mental diets are shown in Tables 1 and 2, respectively. tives (Aviagen, 2016). The CTR diets were formulated to achieve the nutrient specifications of the broiler breeders during rearing and Collected Data, Sampling, and Analytical prebreeder periods as per Ross 308 Parent Stock Nutri- tion Specifications (Aviagen, 2016). Raw materials, Determinations source of insoluble fiber, were included in the FBR diets Body Weight, Uniformity, and Mortality Pullets were to dilute 15% of their AMEn and nutrient content (crude weekly individually weighed, and average BW and BW protein and digestible amino acids, digestible phospho- CV, calculated per pen. Mortality was recorded daily rous and calcium, and vitamin and mineral premix). and accumulated mortality calculated to make compar- Among these, a forage pellet, containing 67% ray grass isons among the different treatments. and 33% barley straw, was used. The AMEn estimated Feed Experimental feed samples were collected from value of the forage pellet was 892 kcal, and its nutritional the different diets supplied during the rearing and pre- analyzed values were as follows: dry matter, 89.7%; CP, breeder periods of the trial. These samples were ground 11.6%; crude fiber (CF), 24.8%; neutral detergent fiber and stored at 5 C until they were analyzed in duplicate. (NDF), 48.1%; acid detergent fiber (ADF), 30.1%; acid Diet proximate analyses were performed in accordance detergent lignin (ADL), 8.1%; crude fat, 1.3%; and with the method of the Association of Official Agricul- ash, 11.7%. In the diets supplemented with vitamin C, tural Chemists (AOAC) International (2005): dry mat- 200 mg/kg of a stabilized (phosphorylated) Na/Ca salt ter (Method 934.01), crude protein (Method 968.06), CF of L-ascorbic acid was included. (Method 962.09), NDF/ADF/ADL (Method 973.18), Feed was provided ad libitum during the first week to crude fat (Method 2,003.05), and ash content (Method ensure a correct initial development. From this moment 942.05). All analyses were carried out in the Servei de on, to follow the same standard BW profile in the 4 treat- Nutricio i Benestar Animal (SNiBA) laboratory of the ments, the total daily feed distributed per pen was UAB. Table 1. Ingredients (%) of the experimental diets. Starter II Grower Prebreeder Ingredients Starter I CTR FBR CTR FBR CTR FBR Wheat 35.2 39.4 - 43.8 - 43.5 - Corn 24.9 24.9 36.9 16.3 22.9 24.8 21.8 Soybean meal 48% 25.2 25.5 10.5 - - 6.3 - Rye - - 12.0 - 28.7 - 33.1 Forage pellet1 - - 5.4 - 16.3 - 11.5 Sunflower 28–30% 5.0 - 15.0 17.0 8.7 12.0 9.7 Wheat bran (high starch) 3.0 5.0 16.1 17.6 20.0 8.0 20.0 Dicalcium phosphate 2.33 2.17 1.70 1.83 1.40 1.53 0.97 Soybean oil 2.00 0.50 - 0.98 - 0.57 - Limestone 0.97 0.97 0.80 0.90 0.63 1.97 1.63 Premix2 0.30 0.50 0.42 0.50 0.42 0.50 0.43 Sodium bicarbonate 0.27 0.31 0.41 0.36 0.40 0.29 0.37 L-lysine sulfate 54.6% 0.24 - - - - - - L-Lysine HCL 65% - 0.19 0.32 0.22 0.16 0.12 0.15 Salt 0.23 0.20 0.05 0.17 0.05 0.22 0.06 DL-Methionine 0.20 0.22 0.16 0.11 0.11 0.11 0.10 Choline Cl-60% 0.10 0.10 0.10 0.10 0.10 0.07 0.08 L-Threonine 0.05 0.09 0.09 0.10 0.06 0.03 0.03 Xylanase 1 ß- glucanase - 0.01 0.01 0.01 0.01 0.01 0.01 Vitamin C mg/kg3 200 Vitamin C mg/kg C14 200 200 200 200 200 200 Vitamin C mg/kg C25 0 0 0 0 0 0 Abbreviations: CTR, control diet; FBR, fibrous diet. 1 Forage pellet composition: Ray grass (67%) and barley straw (33%). 2 Premix provided the following per kilogram of diet: vitamin A (retinyl acetate), 11,000 IU; vitamin D3 (cholecalciferol), 3500 IU; vitamin E (DL-a-tocopheryl acetate), 100 IU; vitamin K3, 3.00 mg; vitamin B1 (thiamin), 3.00 mg; vitamin B2 (riboflavin), 6.00 mg; vitamin B5 (pantothenic acid), 13.00 mg; vitamin B6 (pyridoxine hydrochloride), 4.00 mg; vitamin B12 (cyanocobalamin), 0.02 mg; vitamin B3 (nicotinic acid), 30.00 mg; vitamin B9 (folic acid), 1.50 mg; vitamin B7 (biotin), 0.20 mg; iodine (calcium iodate), 1.25 mg; Mn (manganous oxide), 120.00 mg; Cu (cupric sulfate), 16.00 mg; Zn (zinc oxide), 110 mg; Se (sodium selenite), 0.30 mg; Fe (iron sulphate), 40.00 mg. 3 Vitamin C product source: Rovimix STAY–C 35 (0.0571% inclusion). 4 C1: Diets vitamin C supplemented. 5 C2: Diets not vitamin C supplemented. 2636 ASENSIO ET AL. Table 2. Metabolizable energy and nutrient composition (% as fed-basis, unless otherwise indicated) of the experimental diets. Starter II Grower Prebreeder Item Starter I CTR FBR CTR FBR CTR FBR AMEn, kcal/kg 2,824 2,804 2,399 2,600 2,240 2,700 2,295 Nutrient composition Dry matter1 87.7 88.0 87.8 88.5 87.4 88.4 88.1 Crude protein1 17.9 18.3 15.7 13.1 11.8 13.8 11.8 dig Lys 0.95 0.96 0.81 0.54 0.43 0.54 0.43 dig Met 0.47 0.48 0.40 0.34 0.28 0.33 0.27 dig M 1 C 0.74 0.76 0.63 0.56 0.45 0.56 0.45 dig Thre 0.64 0.67 0.56 0.46 0.37 0.43 0.34 dig Trp 0.20 0.20 0.16 0.14 0.11 0.15 0.11 Crude fiber1 4.5 3.2 8.1 7.2 8.8 5.0 7.5 Neutral detergent fiber1 11.6 10.3 17.6 17.5 19.9 12.2 18.4 Acid detergent fiber1 5.0 3.7 8.2 7.9 9.2 5.2 8.0 Acid detergent lignin1 0.93 0.76 2.42 2.34 2.56 1.60 2.64 Crude fat1 4.2 2.5 2.9 3.2 2.3 2.7 2.4 Ash1 6.4 5.9 6.4 5.7 6.1 6.4 6.5 Ca 1.06 1.00 0.88 0.90 0.78 1.22 1.02 dig P 0.48 0.45 0.40 0.42 0.35 0.36 0.29 Phytate 0.99 0.93 1.30 1.29 1.14 1.08 1.18 Vitamin C, mg/kg1 117.0 Vitamin C, mg/kg C11,2 134.0 164.0 152.0 131.5 150.0 116.5 Vitamin C, mg/kg C21,3 ,5 ,5 ,5 ,5 ,5 ,5 Abbreviations: CTR, control diet; FBR, fibrous diet. 1 Analyzed values. 2 C1: Diets vitamin C supplemented. 3 C2: Diets not vitamin C supplemented. Carcass Traits At 22 wk, after weighing all of the pul- Tibia Parameters Tibias of right legs (2 birds/pen) lets, the 2 closest birds to the average BW of each pen were used to determine tibia length (TL), tibia width were euthanized to perform measurements and take (TW), tibia breaking strength (BS) (maximum load samples to study carcass traits, intestinal morphology, endurance), apparent elastic modulus (EM), and ash and tibia parameters. content. TL was measured from the intercondylar Breast muscle (pectoralis major plus pectoralis mi- eminence to the tip of the lateral malleolus, and TW nor), abdominal fat (surrounding the gizzard and peri- was measured in the narrowest part of the tibia diaph- cloacal), empty digestive tract (from proventriculus to ysis. To perform these 2 measurements, a calliper cloaca, both included), and oviduct were extracted and (0.03-mm precision and 0.01-mm resolution) was used. weighed to calculate their BW percentage. To determine tibia BS and EM, the method of 3-point Intestinal Morphology Tissue samples that were 3-cm flexural bending test (Fleming et al., 1998) was taken long were taken from the jejunum, at the midpoint be- into account; measurements were made by using an tween the bile duct entry and Meckel’s diverticulum (2 MTS Alliance RT/5 material testing system (MTS birds/pen). Samples were fixed in 10% buffered formalin Systems Corp.; Eden Prairie, MN 55344) (force resolu- solution, dehydrated in a graded ethanol series, and tion, 0.001 N and distance resolution, 0.001 mm). These embedded in paraffin. Four cross sections (4-mm-thick studies were carried out at the Institut de Recerca i each) were cut separately from each sample and included Tecnologia Agroalimentaria (Girona, Spain). To deter- in the same preparation. Sections were stained with he- mine the percentage of ash content, right tibias were matoxylin and eosin, with the method of Hampson (1986) boiled and any adherent tissue was cleaned off; they were being taken into account. Histomorphological measure- weighed, dried (109 C 12 h), chemically cleaned (in ments of the intestinal mucosa were carried out: at least acetone for 48 h), dried (109 C 12 h), weighed again, and the height of 30 intact villi (distance from the villus tip to burned in a muffle oven overnight (550 C). These ana- the villus–crypt junction) and the depth of 30 crypts of lyses were carried out in the SNiBA laboratory of the Lieberk€ uhn (depth of the invagination between adjacent UAB. villi) of each preparation were measured, and the ratio Alkaline Phosphatase To determine ALP, the 4 between both was calculated. The measurements were closest pullets to the average BW of each pen were cho- taken from digital photographs of the preparations, which sen at 6 wk and blood was sampled at 6, 15, and 22 wk of were captured in a Leica SCN400 scanner (Leica Micro- age. Blood samples were spun at 1,107 g for 20 min to systems CMS GmbH, Wetzlar, Germany) at 40!. The obtain the serum, which was stored at 220 C until the images obtained were analyzed by the Slide Path Digital moment to be analyzed. To test ALP, a colorimetric Image Hub, version 4.0.7 software (Leica Biosystems method based on catalytic transformation of p-nitrophe- GmbH, Nussloch, Germany). This analysis was carried nylphosphate as substrate (International Federation of out in the Universidad CEU Cardenal Herrera, Valencia, Chemical Chemistry method) on the Beckman Coulter Spain. AU analyzer (Beckman Coulter, Inc.; Brea, CA 92821) FIBROUS DIET AND VITAMIN C IN BREEDERS 2637 was used. ALP analyses were carried out by the Servei de GFP, NFOP, and tail- and wing-feather integrity status, Bioquímica Clínica Veterinaria of the UAB. fiber level (CTR and FBR) and vitamin C inclusion (C2 Behavior At 6, 15, and 22 wk of age, behavior of the and C1) effects were contrasted through a Wilcoxon pullets was observed by using an instantaneous, visual rank test (Mann–Whitney test). The 4 combinations of scan-sampling technique, and the method followed by fiber level and vitamin C inclusion effects (CTR/C2, Hocking et al. (2004) was taken into account. Grasping CTR/C1, FBR/C2, and FBR/C1) were contrasted feather pecking (GFP) (grasping tail and back feathers through a Kruskal–Wallis test; when this test result of other pullets) and non–food object pecking (NFOP) was significant, pairwise combinations were contrasted (pecking at drinkers and pen walls) behaviors were by the Wilcoxon rank test. observed during two 20-min sessions a day, once during the morning (9h.30– 12h.30) and once in the afternoon RESULTS (13h.30– 16h.30). Observations were made per block of 4 pens at 1-min intervals, in a random order, after all Body Weight, Uniformity, and Mortality feeding-related activities had ceased and after allowing 5 min for the birds to adjust to the presence of the Table 3 shows BW and BW CVs at 6, 15, and 22 wk. observer. The number of pullets engaged in each BW and BW CVs were not affected by fiber level or behavioral category was recorded and expressed in pro- vitamin C inclusion, nor was there any interaction portion to the total number of active animals. All ob- (P . 0.05). BW CVs between the pullets fed the CTR servations were performed by the same observer. and FBR diets were similar at 6 wk (11.36 and 11.59%, The integrity of the tail and wing feathers of all of the respectively, P 5 0.818), 15 wk (9.71 and 10.32%, respec- pullets was scored at 22 wk of age. A feather score between tively, P 5 0.449), and 22 wk (8.68 and 9.02%, respec- 0 and 5 was used: (0) fully feathered, (1) rough, (2) some tively, P 5 0.714). Cumulative mortality (%) at 22 wk broken feathers and small bald areas, (3) heavily broken was not affected by fiber level (CTR: 2.22% and FBR: feathers and some bald areas, (4) almost bald/large bald 0.56%, P 5 0.372) or vitamin C inclusion (C2: 0.52% areas, and (5) bald (no feather cover). To perform this and C1: 2.38%, P 5 0.189); the combination of effects scoring, the Protocol for Scoring the Feather Cover of (fiber level/vitamin C inclusion) was not significant Broiler Breeders (Aviagen, 2010) was taken into account. either (P 5 0.240). Statistical Analysis Feed and Nutrient Intake The statistical analysis was performed with the Feed intake between 0 and 22 wk of age is shown in version 3.5.1 R program (R Core Team, 2018). Factorial Figure 1. After weekly adjustments to follow the standard ANOVA was carried out to analyze BW, BW CV, intes- BW profile in all treatments, accumulated feed intake (g) tinal morphology, BS, EM, tibia ash content, TL, and at 22 wk of the FBR treatments was 17.3% higher than TW. The factors were fiber level (CTR and FBR) and that of the CTR (FBR/C2: 12,431 6 9.9 and FBR/C1: vitamin C inclusion (C2 and C1). Mortality was 12,407 6 8.8 vs. CTR/C2: 10,268 6 52.5 and CTR/ analyzed by contrasting fiber level (CTR and FBR), C1: 10,264 6 36.0) (P , 0.01). vitamin C inclusion (C2 and C1), and their combina- The analyzed values of the experimental diets showed tions (CTR/C2, CTR/C1, FBR/C2, and FBR/C1). that, on average, the FBR diets had 12.9% less CP, Several contingency tables were created and solved 8.5% less crude fat, 37.5% more CF, 29.1% more NDF, through a Fisher’s exact test for count data. To analyze 34.7% more ADF, and 38.9% more ADL. Vitamin Table 3. Effects of fiber level and vitamin C inclusion on body weight (BW) and body weight coefficient of variation (CV) at 6, 15, and 22 wk of age. BW (g)1 BW CV (%)2 Effects 6 wk 15 wk 22 wk 6 wk 15 wk 22 wk Fiber level CTR 700 1,831 2,693 11.36 9.71 8.68 FBR 687 1,818 2,684 11.59 10.32 9.02 Vitamin C inclusion C2 695 1,831 2,692 10.56 10.32 9.24 C1 691 1,818 2,685 12.39 9.72 8.46 SEM 5.2 12.0 16.0 0.213 0.572 0.644 P-values Fiber level 0.085 0.460 0.692 0.818 0.449 0.714 Vitamin C inclusion 0.565 0.443 0.804 0.081 0.461 0.393 Interaction 0.309 0.892 0.730 0.281 0.180 0.258 Abbreviations: C2, not vitamin C–supplemented diet; C1, vitamin C–supplemented diet; CTR, control diet; FBR, fibrous diet. P , 0.05 was considered significant. 1 n 5 192 for fiber level and vitamin C effects and n 5 96 for the interaction. 2 n 5 16 for fiber level and vitamin C effects and n 5 8 for the interaction. 2638 ASENSIO ET AL. 120 110 100 90 Feed intake (g/bird/day) 80 70 60 50 40 30 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Age (weeks) CTR/C+ CTR/C- FBR/C+ FBR/C- Figure 1. Weekly feed intake of the broiler breeder pullets from 0 to 22 wk of age, depending on fiber level and vitamin C inclusion. Values are pooled means of 8 replicates per treatment. CTR/C2: control diet not vitamin C supplemented, CTR/C1: control diet vitamin C supplemented, FBR/C2: fibrous diet not vitamin C supplemented, and FBR/C1: fibrous diet vitamin C supplemented. Pooled SEM 5 5.01. C–supplemented diets of the treatments CTR/C1 and 2.05%, P 5 0.136) or for oviduct (12.5 g and 16.2 g/10 kg FBR/C1 presented an average vitamin C concentration BW, P 5 0.227). (mg/kg) of 138 6 18.2, whereas in the nonsupplemented di- ets of the treatments, CTR/C2 and FBR/C2, vitamin C Intestinal Morphology concentration (mg/kg) was ,5 (mg/kg). Histomorphological parameters of the intestinal mu- cosa of the middle portion of the jejunum are shown in Carcass Traits Table 5. At 22 wk of age, there were neither fiber level nor vitamin C inclusion effects on the intestinal The effects of fiber level and vitamin C inclusion on morphology: villus height, Lieberk€ uhn crypt depth, and carcass traits (relative to BW) at 22 wk of age are shown their ratio. in Table 4. There was no vitamin C inclusion effect, but pullets fed the FBR diet, compared with those fed the CTR diet, had less breast muscle (18.5 vs. 19.8%, Tibia Parameters P 5 0.001) and higher abdominal fat deposition (1.14 The effects of fiber level and vitamin C inclusion on vs. 0.87%, P 5 0.031), total empty digestive tract tibia BS, EM, and ash content at 22 wk are shown in (5.07 vs. 4.63%, P 5 0.001), proventriculus plus gizzard Table 6. There was no vitamin C inclusion effect, but (2.04 vs. 1.94%, P 5 0.035), and caecum plus rectum tibias from the pullets fed the CTR diet, compared (0.90 vs. 0.64%, P , 0.001). However, there was no sig- with those fed the FBR diet, were able to endure more nificant difference between the pullets fed the FBR diet weight before breaking, their BS values were higher and those fed the CTR diet, for small intestine (2.13 and (45.1 vs. 41.4 kgf/mm2, P 5 0.009), they were also Table 4. Effects of fiber level and vitamin C inclusion on carcass traits (% BW, oviduct: g/10 Kg BW) at 22 wk of age. Carcass traits Effects Breast muscle Abdominal fat Total digestive tract Proventriculus 1 gizzard Small intestine Caecum 1 rectum Oviduct Fiber level CTR 19.8 0.87 4.63 1.94 2.05 0.64 16.2 FBR 18.5 1.14 5.07 2.04 2.13 0.90 12.5 Vitamin C inclusion C2 19.0 1.03 4.88 1.97 2.12 0.79 13.4 C1 19.3 0.98 4.81 2.02 2.08 0.74 15.3 SEM (n 5 32) 0.23 0.082 0.081 0.033 0.042 0.041 2.12 P-values Fiber level 0.001 0.031 0.001 0.035 0.136 ,0.001 0.227 Vitamin C inclusion 0.345 0.601 0.526 0.368 0.265 0.343 0.536 Interaction 0.595 0.105 0.897 0.530 0.365 0.895 0.439 Abbreviations: C2, not vitamin C–supplemented diet; C1, vitamin C–supplemented diet; CTR, control diet; FBR, fibrous diet. P , 0.05 was considered significant. FIBROUS DIET AND VITAMIN C IN BREEDERS 2639 Table 5. Effects of fiber level and vitamin C inclusion on the histomorphological parameters of the intestinal mucosa of the middle portion of the jejunum at 22 wk of age. Effects Villi height (mm) Lieberk€ uhn crypt depth (mm) Ratio height/depth Fiber level CTR 974 160 6.12 FBR 935 166 5.67 Vitamin C inclusion C2 982 162 6.00 C1 925 165 5.73 SEM (n 5 21) 32.6 3.5 0.250 P-values Fiber level 0.471 0.411 0.323 Vitamin C inclusion 0.318 0.618 0.570 Interaction 0.955 0.884 0.905 Abbreviations: C2, not vitamin C–supplemented diet; C1, vitamin C–supplemented diet; CTR, control diet; FBR, fibrous diet. P , 0.05 was considered significant. more rigid or less ductile, their EM values were higher At 6 wk, pullets did not perform GFP at all, and there (43.3 vs. 36.4 kgf/mm, P 5 0.001), and they had higher were no fiber level or vitamin C inclusion effects on ash content (41.8 vs. 40.0%, P 5 0.001). NFOP. At 15 wk, the combinations of fiber level and Regarding tibia measurements, no effects of fiber level vitamin C effects showed a significantly higher percentage or vitamin C inclusion were found. Pullets fed the CTR of GFP in the CTR/C2 treatment than in the other treat- diet, compared with those fed the FBR diet, had no sig- ments (P 5 0.009); there were no significant differences nificant differences in TL and TW values (TL: 130 mm among the CTR/C1, FBR/C2, and FBR/C1 treat- and 131 mm, P 5 0.147; TW: 7.01 mm and 7.09 mm, ments. Regarding NFOP, the CTR/C1 treatment had a P 5 0.277). significantly higher percentage than the FBR/C1 treat- ment (P 5 0.043); the CTR/C2 and FBR/C2 treatments Alkaline Phosphatase had intermediate values. At 22 wk, and for GFP and NFOP, the combination of effects showed no significant The effects of fiber level and vitamin C inclusion on differences between CTR/C2 and CTR/C1, nor between ALP serological levels at 6, 15, and 22 wk are shown in FBR/C2 and FBR/C1 treatments; nevertheless, the per- Table 7. At 6 and 22 wk, there was no vitamin C inclusion centage values of the CTR treatments were significantly effect, but pullets fed the CTR diet, compared with those higher than were those of the FBR treatments (GFP: fed the FBR diet, had higher ALP levels (6 wk: 10,045 vs. P 5 0.029; NFOP: P 5 0.003). 8,042 IU, P 5 0.001; 22 wk: 1,107 vs. 775 IU, P , 0.001). Tail-feather score results at 22 wk showed a significantly However, at 15 wk, there was neither fiber level nor higher value of the CTR/C2 treatment than with other vitamin C inclusion effect on serum ALP levels. treatments (P , 0.001); there were no significant differ- ences among the CTR/C1, FBR/C2, and FBR/C1 Behavior treatments. Wing-feather score results showed that the CTR/C2 treatment had a significantly higher value at The effects of fiber level and vitamin C inclusion on 22 wk (P , 0.001). The CTR/C1 treatment had an inter- bird behavior (GFP and NFOP) at 6, 15, and 22 wk mediate value significantly higher than did those of the and on tail- and wing-feather scores at 22 wk of age FBR/C2 and FBR/C1 treatments, which showed no dif- are shown in Table 8. ference between them. Table 6. Effects of fiber level and vitamin C inclusion on tibia breaking strength (BS), elastic modulus (EM), and ash content at 22 wk of age. Effects BS (kgf/mm2) EM (kgf/mm) Ash content (%) Fiber level CTR 45.1 43.3 41.8 FBR 41.4 36.4 40.0 Vitamin C inclusion C2 43.5 40.7 41.2 C1 43.0 39.0 40.6 SEM (n 5 32) 0.97 1.26 0.37 P-values Fiber level 0.009 0.001 0.001 Vitamin C inclusion 0.732 0.351 0.240 Interaction 0.988 0.632 0.760 Abbreviations: C2, not vitamin C–supplemented diet; C1, vitamin C– supplemented diet; CTR, control diet; FBR, fibrous diet. P , 0.05 was considered significant. 2640 ASENSIO ET AL. Table 7. Effects of fiber level and vitamin C inclusion on alkaline Lower breast muscle mass of the birds fed the FBR diet phosphatase (ALP) serological level at 6, 15, and 22 wk of age. might be related to its higher insoluble fiber inclusion. ALP (IU) Kluth and Rodehutscord (2009) studied the inevitable Effects 6 wk 15 wk 22 wk endogenous amino acid loss at the terminal ileum of broilers fed diets with 2 different insoluble fiber (cellu- Fiber level lose) levels. The results showed a significant increase in CTR 10,045 1,371 1,107 FBR 8,042 1,344 775 CP and amino acid flow at the terminal ileum when the Vitamin C inclusion cellulose level in the diet was increased. In another study C2 8,989 1,408 987 carried out with roosters force-fed N-free diets, Parsons C1 9,098 1,304 890 SEM (n 5 64) 424.5 61.4 41.7 et al. (1983) found that amino acid excretion was higher P-values when high-insoluble fiber diet (with 17.5% cellulose) was Fiber level 0.001 0.765 ,0.001 used, in comparison with a low-insoluble fiber diet. As Vitamin C inclusion 0.856 0.247 0.114 Interaction 0.058 0.104 0.220 noted by de Lange et al. (1989), the effect of cellulose might be mediated by the mechanical effect of fiber. Like- Abbreviations: C2, not vitamin C–supplemented diet; C1, vitamin C– supplemented diet; CTR, control diet; FBR, fibrous diet. wise, Leterme et al. (1998) reported that fiber has an P , 0.05 was considered significant. abrasive effect on the intestinal wall and may result in an increase in endogenous cell losses and nutrients to DISCUSSION the lumen. Therefore, the birds fed the FBR diet might have had higher amino acid endogenous losses, and this Effect of Fibrous Diet might impair breast muscle deposition. Furthermore, In the present study, it was expected that feeding the more developed digestive tract of the birds fed the broiler breeder pullets with FBR diets, which increased FBR diet found in this study might also be a cause of their feed allocation and was supposed to lessen competition, less developed breast muscle mass because amino acid might result in higher BW uniformity and lower mortal- allocation for the development and turnover of this ity. Opposite to our expectations, the FBR diet did not higher digestive tract might have been to the detriment improve uniformity or reduce mortality. These results of breast muscle growth. are congruent with those of other authors who fed broiler The relevance of phytate–protein complexes should breeder pullets with diluted diets in rearing and found also be highlighted in lowering protein utilization in neither uniformity improvement (Zuidhof et al., 2015; monogastric animals. In the present study, diets were de Los Mozos et al., 2017) nor mortality reduction (de not phytase supplemented, and pullets fed the FBR Los Mozos et al., 2017). It would be interesting to study diet consumed 19.2% more phytate throughout rearing the effect of FBR diets on these parameters in field con- and prebreeder periods (up to 22 wk of age) than did ditions, where management is poorer and, thus, compe- those of the control (FBR diet: 146 g vs. control: 118 tition is higher. g, P , 0.01). Phytate levels were not analyzed but are Table 8. Effects of fiber level and vitamin C inclusion on the percentage of birds performing grasping feather pecking (GFP) and non–food object pecking (NFOP) at 6, 15, and 22 wk and on tail- and wing-feather score at 22 wk of age. GFP1 NFOP1 Tail-feather score2 Wing-feather score2 Effects 6 wk 15 wk 22 wk 6 wk 15 wk 22 wk 22 wk 22 wk Fiber level CTR 0 7.74 5.17 12.4 24.0 35.1 0.73 1.12 FBR 0 0.29 0.05 12.9 14.1 18.5 0.20 0.70 Vitamin C inclusion C2 0 5.83 3.73 13.4 18.4 28.2 0.66 1.01 C1 0 2.20 1.49 11.9 19.7 25.4 0.25 0.80 Fiber level/vitamin C CTR/C2 0 11.53a 7.47a 14.6 22.4a,b 35.2a 1.15a 1.26a CTR/C1 0 3.96b 2.88a 10.2 25.7a 35.1a 0.30b 0.98b FBR/C2 0 0.14b 0b 12.2 14.4a,b 21.2b 0.19b 0.76c FBR/C1 0 0.45b 0.10b 13.6 13.7b 15.8b 0.21b 0.63c P-values Fiber level 0 0.016 0.008 0.773 0.005 ,0.001 ,0.001 ,0.001 Vitamin C inclusion 0 0.104 0.828 0.707 0.778 0.537 ,0.001 0.029 Fiber level/vitamin C 0 0.009 0.029 0.924 0.043 0.003 ,0.001 ,0.001 a-c Means within a column and within a source with no common superscript differ significantly. Abbreviations: CTR, control diet; C2, not vitamin C–supplemented diet; C1, vitamin C–supplemented diet; FBR, fibrous diet. Treatments: CTR/C2: control diet not vitamin C supplemented, CTR/C1: control diet vitamin C supplemented, FBR/C2: fibrous diet not vitamin C supplemented, and FBR/C1: fibrous diet vitamin C supplemented. Tail- and wing-feather scores: (0) fully feathered, (1) rough, (2) some broken feathers and small bald areas, (3) heavily broken feathers and some bald areas, (4) almost bald/large bald areas, and (5) bald (no feather cover). 1 n 5 32 for fiber level and vitamin C effects and n 5 16 for the combinations fiber level/vitamin C. 2 n 5 192 for fiber level and vitamin C effects and n 5 96 for the combinations fiber level/vitamin C. FIBROUS DIET AND VITAMIN C IN BREEDERS 2641 based on formulation levels. The study carried out with diets with insoluble fiber and lignin (lignocellulose) broilers by Ravindran et al. (2001) showed a linear improved femur BS and femur and tibia ash weights. BS improvement in ileal digestibility of nitrogen and all represents maximum load endurance: less mineralized amino acids after the addition of increasing levels of sup- bones have lower values; EM reflects the intrinsic stiffness plemental phytase to a lysine-deficient diet. Regarding or rigidity of the bone: less mineralized bones also have lysine, its percentages of improvement in digestibility lower values (Turner and Burr, 1993); and because bone with 500 and 1000 FTU/kg were 4.5 and 5.9%, mineralization provides compressional strength to bone, respectively. the bone ash content has been used as an indicator of The higher amino acid endogenous losses and require- bone strength (Rath et al., 2000). The results of the pre- ments to develop and maintain the digestive tract might sent study suggest that skeletal strength of the pullets be related to the more important abdominal fat deposi- fed the FBR diet is poorer and thus mineral absorption tion found in the birds fed the diluted diet. Yalçin might have been impaired. This would not be related to et al. (2010) found that feeding broiler chickens diets changes in the intestinal morphology produced by the containing 19.2, 16.6, and 15.5% CP (low protein) led FBR diet because this research did not show effects of fiber to an increase in the total-carcass fat deposition, as level on villus height, Lieberk€ uhn crypt depth, and their compared with chickens fed diets containing 22.9, 19.9, ratio. These results are in agreement with those from and 18.2% CP in the starter, grower, and finisher phases, some broiler studies which have not reported a relation- respectively. Corzo et al. (2006) and Zhan et al. (2006) ship between intestinal morphology parameters and the reported significant increases in the abdominal fat per- presence of structural components (raw materials source centage after feeding chickens methionine-deficient di- of fiber or whole cereals) in broiler diets (Wu et al., 2004; ets. Berri et al. (2008) and Nasr and Kheiri (2011) Wu and Ravindran, 2004; Husveth et al., 2015). showed that dietary L-lysine supplementation lowered However, high-insoluble fiber inclusion might have accel- fat deposition in broiler chickens. In fact, lysine pro- erated the passage time of the digesta through the diges- motes lean meat production by reducing carcass fatness tive tract and reduce Ca and P absorption. The study by via lipogenesis inhibition (Grisoni et al., 1991). Enting et al. (2007a) compared a normal-density diet Nowadays, the target is to control an excess of breast with low-density diets in broiler breeders at 22 wk. They muscle and to stimulate abdominal fat deposition before reported a shorter mean retention time of the digesta in the onset of production because fat content in the rear- the jejunum and ileum, the difference being significant be- ing period is related to egg persistence in the second tween normal- and low-density diets in the jejunum. In half of the production phase (van Emous et al., 2015). accordance with the results of the study by Leeson et al. Therefore, the fact of having the pullets fed the FBR (1991), the shorter retention time seems to be related to diet, with less breast muscle and more abdominal fat, an increase in the insoluble fiber content of the low- is an important advantage of this feed strategy. density diets. The less time the digesta spend in the The higher development of the proventriculus plus jejunum and ileum is negatively related to the amount of gizzard of the birds fed the FBR diet might be related Ca and P absorbed by paracellular absorption, a passive to the high lignin content of most insoluble fiber sources, and nonsaturable way (Fujita et al., 2008; Adedokun which leads to a longer retention of the feed in the and Adeola, 2013), which, in turn, may impair bone gizzard, improving its muscular development and thus mineral deposition and skeletal strength. Likewise, it its function (Hetland and Svihus, 2001; Hetland et al., should be highlighted that the formation of Ca–phytate 2003; Jimenez-Moreno et al., 2010). Likewise, Sacranie complexes reduces phytate hydrolysis and Ca and phos- et al. (2012) studied broiler chickens exposed to FBR di- phorus digestibility (Sebastian et al., 1996; Tamim et al., ets diluted with coarse hulls or the same hulls finely 2004; Plumstead et al., 2008), and, as was explained ground. The large particle size of the coarse hulls and before, in the present study, pullets fed the FBR diet their hardness as a result of the insoluble fiber content consumed a higher, significant amount of phytate than explained why birds consuming the coarse hull diet did those fed the CTR diet. Future research should developed the heaviest gizzards. However, birds fed the consider the impact on bone mineralization of phytate fine hull diet also exhibited heavier gizzards than did intake in FBR diets. the control group, although less so than those of the There are no studies that have used ALP to assess coarse hull group. skeletal development (bone mineral deposition) in In the present study, it was hypothesized that there broiler breeder pullets in rearing. However, in chickens, would be greater skeletal strength in the broiler breeder elevated ALP activity has been predominantly related fed the FBR diet than in the control pullets. However, to increased osteoblastic activity and used as a marker the pullets fed the diluted diet had tibias with significantly for evaluating skeletal health and bone disease, such as lower BS, EM, and ash content. These results might signal skeletal growth, rickets, fracture repair, and osteomye- poor bone mineral deposition early in rearing because it is litis (Lumeij and Westerhof, 1987). More recently, stated that 90% of the skeleton of the broiler breeders is Ekmay et al. (2012) used ALP serological levels to assess already developed at 11–13 wk of age (Ross Parent Stock broiler breeder growth and bone mineral deposition dur- Management Handbook, Aviagen, 2013). Nevertheless, ing transition into sexual maturity (24–26 wk of age). In results from this study are not congruent with those of the present study, pullets fed the FBR diet had lower Oikeh et al. (2019), who found that broilers fed diluted ALP serological levels at 6 and 22 wk, which suggests 2642 ASENSIO ET AL. that their bone mineral deposition during the starter and supplement 200 mg/kg of vitamin C (after Whitehead prebreeder periods was poorer than that of the control. It et al. (1993) recommendation), but the average analyzed has to be emphasized that the ALP serological level is value of the different diets was 138 mg/kg. In a similar directly related to bone development and mineral depo- way, in the present study, the premix provided an sition; thus, it is a better indicator of skeletal develop- adequate level of vitamin D3, which might have shaded ment than is BW, which is an indirect indicator. the effect of vitamin C on the enhancement of 1,25-D In this study, the FBR diet did not affect bone growth, production. Therefore, higher levels of vitamin C supple- as TL and TW were not influenced. However, ALP sero- mentation and a vitamin D3–deficient diet might be op- logical levels at 6 wk and BS, EM, and ash content at 22 tions for the experimental design of a future study. wk of age signal poorer bone mineral deposition of the Another reason to study the vitamin C effect on skeletal birds fed the FBR diet (lower ALP serum values and strength was the fact that its endogenous synthesis in lower BS and EM parameters and ash content of the poultry may not be sufficient during stress periods, tibias). Therefore, the FBR diet did not influence skel- when the requirements may exceed the synthesizing abil- etal growth; nonetheless, mineral deposition was ity (Gous and Morris, 2005). However, in experimental affected. conditions, the management is correct, and thus, the In the present study, it was hypothesized that there stress is low. It would be interesting to study the vitamin would be lower stereotypic behaviors in the broiler C effect in field conditions, where management is poorer breeders fed the FBR diet than in the control pullets. and thus competition and stress are higher. As we expected, pullets fed the diluted diet showed a In this study, it was hypothesized that there would be reduction in GFP and NFOP, stereotypic behaviors, at lower stereotypic behaviors in vitamin C–supplemented 22 wk of age. The reduction of GFP and NFOP in the broiler breeders. At 15 wk, vitamin C inclusion reduced birds fed the FBR diet may be explained by the GFP of the pullets fed the CTR diet (lower in feed allow- increasing satiety coming from its insoluble fiber con- ance) to the same level as the FBR diet did. As was tent. Oat hulls, a source of insoluble fiber, have been explained previously, vitamin C recovery was lower used in several studies to dilute the energy of the diets than expected, and in experimental conditions, the birds (Zuidhof et al., 1995; Hocking et al., 2004; Sandilands may be little subjected to stress; these may also be the et al., 2005, 2006; Hocking, 2006; Enting et al., 2007a, causes that vitamin C supplementation did not show a b; Nielsen et al., 2011); however, this compound was more important effect on reducing stereotypic behaviors. primarily designed to increase gastric satiety through Nevertheless, a better feather-cover integrity was found; delaying crop and gizzard emptying. Other studies vitamin C reduced tail- and wing-feather scores of the showed a reduction in stereotypic behaviors in broiler pullets fed the CTR diet, in the case of tail score at the breeders fed FBR diluted diets (Zuidhof et al., 1995; de same level as in the FBR diet. In fact, studies show Jong et al., 2005). Hocking et al. (2004) observed that vitamin C might help birds to cope with stressful decreased spot pecking in broiler breeders fed diets situations (Satterlee et al., 1993, 1994; Jones et al., with 20% oat hulls and 5% sugar beet pulp. 1996, 1999). It has to be highlighted that feather cover The wing-feather score at 22 wk of the pullets fed the protects female broiler breeders from being naturally FBR diet was lower than in those fed the CTR diet. damaged by the males during mating; therefore, it is Therefore, the results of the present study show, at 22 important that the females finish the rearing period wk, a reduction of GFP and at the same time a lower with good feather-cover integrity to obtain correct wing-feather score of the birds fed the FBR diet; there hatchability during production. might be a positive correlation to be further studied. Effect of Vitamin C CONCLUSIONS In the present study, it was hypothesized that a posi- Fibrous diets, formulated with raw material source of tive effect of vitamin C might be obtained on the skeletal insoluble fiber, do not modify broiler breeder pullet uni- strength of broiler breeder pullets. However, vitamin C formity or mortality. Pullets fed the FBR diet have less supplementation did not affect tibia BS, EM, or ash con- breast muscle and more abdominal fat, which is a great tent. Our expectations were based on vitamin C involve- advantage for later production; at 22 wk, their GFP ment in the normal proliferation and differentiation of and NFOP behaviors are lower and their wing feather chondrocytes (essential for the initial formation of integrity is better, which means they undergo less stress; bone and later remodeling) and on the fact that its defi- however, even with their skeletal growth not being modi- ciency results in widespread connective tissue abnormal- fied, their bone mineral content and thus skeletal ities coming from a disruption in collagen synthesis strength are lower. It can be concluded that the FBR (Whitehead and Keller, 2003). In a similar manner, diet improves carcass traits and reduces stereotypic be- Weiser et al., 1988 reported the enhancement of 1,25-D haviors. Vitamin C inclusion does not influence skeletal production in vitamin C–supplemented chicks. One of strength, but it is effective in improving tail- and wing- the possible causes of not obtaining any vitamin C effect feather integrity of the pullets fed the CTR diets, lower on skeletal strength might have been its low inclusion in in feed allowance. Moreover, ALP could be a direct way the feed of our experiment. It was designed to to assess bone mineral deposition, and the tail- and wing- FIBROUS DIET AND VITAMIN C IN BREEDERS 2643 feather scores could be noninvasive practical tests to Fujita, H., K. Sugimoto, S. Inatomi, T. Maeda, M. Osanai, quickly assess stress in rearing farms. Y. Uchiyama, Y. Yamamoto, T. Wada, T. Kojima, H. Yokozaki, T. Yamashita, S. Kato, N. Sawada, and H. Chiba. 2008. Tight junction proteins claudin-2 and -12 are critical for vitamin D- dependent Ca21 absorption between enterocytes. Mol. Biol. Cell ACKNOWLEDGMENTS 19:1912–1921. Gonz alez-Alvarado, J. M., E. Jimenez-Moreno, R. L azaro, and All co-authors participated equally in the research and G. G. 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