Productive performance, bone characteristics, and intestinal morphology of laying hens fed diets formulated with L-glutamic acid

Productive performance, bone characteristics, and intestinal morphology of laying hens fed diets formulated with L-glutamic acid A. P. Pereira,∗ A. E. Murakami,∗ C. Stefanello,† L. C. V. Iwaki,‡ and T. C. Santos∗,1 Department of Animal Science, Universidade Estadual de Maring´ a, Av. Colombo 5790, Maring´ a, PR 87020–900, Brazil; † Department of Animal Science, Universidade Federal do Rio Grande do Sul, Av. Bento Gon¸calves, 7712, Porto Alegre, RS 91540-000, Brazil; and ‡ Department of Odontology, Universidade Estadual de Maring´ a, Av. Mandacaru, 1550, CEP 87.080-000, Maring´ a, PR 87020–900, Brazil ABSTRACT This study was conducted to evaluate the effects of L-glutamic acid (Glu) on the productive performance, eggshell quality, bone characteristics, and morphologic parameters of laying hens. Two hundred and forty 53-wk-old Hy-Line W36 laying hens were randomly allocated into 5 treatments, 6 replicates, and 8 birds each. The experimental period was 16 wk. The treatments consisted of a Basal diet (Glu calculated at 2.68%) and 4 L-glutamic acid inclusion levels: 2.88, 3.08, 3.28, and 3.48%. Glu inclusion levels in the hens diet had an increasing linear effect (P < 0.05) on eggshell Ca concentration and bone (tibiotarsus) strength index, and a decreasing linear effect (P < 0.05) on the Seedor index. Serum biochemistry results for total and ionic Ca, inorganic P, and alkaline phosphatase were affected by blood collection time (04:00 Pm, 03:00 Am, and 12:00 Pm) but not by the treatments. Jejunum morphometric variables were not influenced by

the treatments except for crypt depth, which demonstrated a quadratic effect (P < 0.05). However, proliferating cell nuclear antigen qualitative immunohistochemical analysis of the jejunum showed more positive nuclei in the villus with the addition of Glu. Both with the basal diet and the lowest Glu inclusion treatment, positive nuclei were observed in the crypts and at the base of the villus, while in the treatments with higher Glu levels, positive cells were common all along the villus mucosa, including its extremity. In conclusion, diets formulated with increased levels of Glu had beneficial effects on eggshell Ca concentration, tibiotarsus structure, and proliferative activity of the jejunum of 69wk-old laying hens. Therefore, dietary L-glutamic acid can be considered an alternative additive to improve bone characteristics in the productive phase of laying hens.

Key words: calcium, eggshell, jejunum, PCNA 2019 Poultry Science 98:2500–2508 http://dx.doi.org/10.3382/ps/pey595

INTRODUCTION Glutamate or L-glutamic acid (Glu) is a nonessential amino acid that is converted into glutamine (Gln) through the action of enzyme glutamine synthetase. This amino acid plays a fundamental role in intestinal health, since it is a mandatory precursor for Gln synthesis (D’Mello, 2003). The effects of Glu on the maintenance and improvement of intestinal quality have been well described. In the intestine, Glu works as an oxidative fuel that plays an important role in improving intestinal function, especially through high cell turnover rate of the intestinal mucosa (Burrin and Stoll, 2009). Moreover, it has also been considered as the most important substrate for the development of the intestinal tract (Reeds et al.,

 C 2019 Poultry Science Association Inc. Received May 23, 2017. Accepted December 26, 2018. 1 Corresponding author: [email protected]

2000). The addition of Glu in the diet has demonstrated beneficial effects on the intestinal villus morphology of broilers (Maiorka et al., 2000). The relationship between Glu and bone disorders in humans, and bone metabolism in general, has been largely studied. L-glutamic acid enters the cells via inotropic and metabotropic receptors. Later, receptor and transporter subtypes were described in the osteoblasts and osteoclasts of humans and rodents (Hinoi et al., 2004). Glutamate receptors were also described in other tissues such as the adrenal medulla, liver, pancreatic islets, β -cells, megakaryocytes, keratinocytes, heart, and bones (Yoneda and Ogita, 1986; Skerry and Genever, 2001). This amino acid can act as a paracrine and autocrine endogenous factor to control bone cell activity (Mason et al., 1997), such as the modulation of hydroxyapatite crystals in bone tissues (Fisher et al., 1983; Hunter and Goldberg, 1994). Glutamic acid is present in the majority of feeds, either in a free form or linked to peptides or proteins, and plays a central role in protein metabolism. This

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L-GLUTAMIC ACID FOR LAYING HENS

MATERIAL AND METHODS All procedures used in the current study were approved by the Ethics and Research Committee of the State University of Maring´ a, Maring´ a, Brazil (n◦ 052/2011).

Birds Husbandry Two hundred forty Hy-Line W36 laying hens were randomly allocated into metallic cages (1 m × 0.50 m), in 4 subunits with 8 hens each. The experimental unit was the cage with 8 hens. Feed and water were provided ad libitum, and birds’ weight and egg production were standardized before starting the experiment, i.e., birds were housed with mean weight within an SD of 5% of the mean weight of all birds. Only egg-producing laying hens were housed. The birds were fed with the experimental diets between 53 and 69 wk of age and were subjected to a 14-D adjustment to the experimental diets. The animals were subjected to a 17-h/D lighting program. Average minimum and maximum tempera-

tures, monitored on a daily basis inside the shed, were 18.9 and 30.1◦ C, respectively, and mean air humidity was 64.5%.

Experimental Diets The experimental design was completely randomized with 5 treatments, 6 replicates, and 8 birds per experimental unit. The treatments consisted of 1 basal diet (control, with an estimated Glu value of 1.49% based on the feed composition recommended by Rostagno et al. 2011) and 4 experimental diets formulated with increasing levels of L-glutamic acid (0.2, 0.4, 0.6, and 0.8%) to the basal diet. The experimental diets were formulated based on the feed composition requirements recommended by Rostagno et al. (2011). The ingredients and calculated compositions of the experimental diets are shown in Table 1.

Egg Production and Eggshell Quality Daily feed intake (g/bird per D), feed conversion (kg of feed/dozen eggs and kg of feed/kg of eggs), egg production (%), and egg loss (%) were recorded in 4 cycles of 28 D. Daily egg mass was calculated by multiplying the laying rate (%) by the average weight of eggs (g) divided by 100. The means for each variable was considered as the sum of all eggs (n = 8 birds). At the end of each 28-D period, the following parameters were assessed for 3 consecutive days: average egg weight, albumen height, specific weight, percentage, and shell thickness. All intact eggs from each experimental unit were identified and weighed individually in a precision scale (0.01 g) and submitted to specific weight test using the flotation method in saline solution. Six saline solutions were prepared, ranging from 1.070 to 1.090 g/cm3 with a variation of 0.004 g/cm3 for each solution. Saline solution densities were measured with densimeter oil. After the test, a sample of 3 eggs per experimental unit was used to determine albumen height. Measurements in millimeters (mm) were related to egg weight to determine the Haugh unit: HU log 100 (H + 7.57 – 1.7 W0.37 ), in which H = albumen height (mm) and W = egg weight (g). Shells were washed and dried at room temperature for 72 h, and weighed using a precision digital scale (0.001 g). Shell mass was obtained by dividing dry shell weight by total egg weight. After weighing the shells, shell thickness was measured at 3 points in the central region of each shell using a digital micrometer (Mitutoyo Sul Americana, S˜ ao Paulo, Brazil). Three intact eggs per experimental unit (n = 90) were used on day 28 of each cycle for the measurement of shell strength (kgf), which was obtained with a texturometer TA.XT2i Texture Analyzer with a 29 WarnerBratzler Shear Force probe (Texture Technologies Corp. and Stable Micro Systems Ltd., Hamilton, MA).

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molecule can donate its amino group via transamination for the synthesis of other amino acids, or it can lose its amino via deamination to 2-oxoglutarate (Newsholme et al., 2003). In broilers, Glu has been studied with the objective to improve bone structure of growing broiler chickens. It is considered an efficient source of non-specific nitrogen for the promotion of bone development in broilers, and to reduce the incidence of leg abnormalities, when supplied at high levels in diets (Moraes et al., 1984; Ribeiro et al., 1995, Silva et al., 2001a,b). Housing egg-laying hens in cages provides production optimization as well as feed security. Although egg production is not restricted to laying cages in some countries, this is the main method used in Brazil. More productive and premature strains are being continuously developed, and problems related to bone metabolism continue to be an important concern in the egg-production chain. Some problems are related to the progressive decrease in the mineralized structure of bones, which has been related to limited movement, higher production, and longer productive cycles (Whitehead and Fleming, 2000; Whitehead, 2004). This condition can result in fatigue and osteoporosis, causing suffering to the birds and decreased production. It has been shown that 29% of hens present bone fractures in the legs before they are slaughtered and towards the end of their production cycle (Gregory and Wilkins, 1989). Based on studies describing the effects of glutamic acid on bone characteristics and the intestinal mucosa, and to further our understanding on laying hens physiology, the objective of this study was to evaluate the effects of diets formulated with increasing levels of L-glutamic acid fed to laying hens on egg production as well as bone, blood, and intestinal parameters.

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Table 1. Ingredients and calculated composition analysis of the experimental diets. Dietary L-glutamic acid % Item

Calculated composition, % or as shown ME, kcal/kg Crude protein (CP) Calcium Available phosphorus Sodium Linoleic acid Digestible Lys Digestible Met + Cys Digestible Thr Digestible Trp Digestible Val Glutamic acid Calculated Glutamine Calculated

0.2

0.4

0.6

0.8

58.15 26.23 1.10 9.68 2.55 0.285 0.352 0.274 0.075 0.037 0.013 0.285 0.00 1.00 0.01

58.15 26.23 1.10 9.68 2.55 0.285 0.352 0.274 0.075 0.037 0.013 0.285 0.20 0.80 0.01

58.15 26.23 1.10 9.68 2.55 0.285 0.352 0.274 0.075 0.037 0.013 0.285 0.40 0.60 0.01

58.15 26.23 1.10 9.68 2.55 0.285 0.352 0.274 0.075 0.037 0.013 0.285 0.60 0.40 0.01

58.15 26.23 1.10 9.68 2.55 0.285 0.352 0.274 0.075 0.037 0.013 0.285 0.80 0.20 0.01

2,800 16.50 4.02 0.30 0.23 2.68 0.80 0.73 0.61 0.18 0.76 1.49 1.574

2,800 16.82 4.02 0.30 0.23 2.68 0.80 0.73 0.61 0.18 0.76 1.687 1.574

2,800 16.93 4.02 0.30 0.23 2.68 0.80 0.73 0.61 0.18 0.76 1.884 1.574

2,800 17.05 4.02 0.30 0.23 2.68 0.80 0.73 0.61 0.18 0.76 2.081 1.574

2,800 17.16 4.02 0.30 0.23 2.68 0.80 0.73 0.61 0.18 0.76 2.278 1.574

1 Mineral and vitamin supplement, Nucleopar Animal Nutrition Ltda. (content per kg of diet): vitamin A, 2,550 IU/g; vitamin E, 2,083.33 mg; vitamin D3 , 500 IU/g; vitamin K3 , 650 mg; vitamin B1 , 408.33 mg; vitamin B12 , 2,500 μ g; vitamin B2 , 1,000 mg; vitamin B6 , 412.5 mg; Ac. folic, 66.67 mg; biotin, 8.33 mg; choline, 70,000 mg; Ac. pantothenic, 2,375 mg; methionine, 226,875 mg; niacin, 5,308.33 mg; iron, 12,500 mg; iodine, 258.33 mg; selenium 75 mg; cobalt, 83.33 mg; antioxidant, 1,250 mg. 2 BHT, butyrate hydroxy toluene.

Blood and Bone Analysis Blood serum analysis was performed on the last day of the experiment (69 wk of age). To schedule the blood serum collection, we observed hens and estimated the period after last egg posture to determine the phase of next egg formation. Blood from the ulnar vein (5 mL) was collected from 1 hen per experimental unit (n = 30) at 4:00 Pm (beginning of eggshell formation - 7 h after last egg posture), at 03:00 Am (eggshell production 18 h after last egg posture), and at 12:00 Pm (empty uterus with egg in magnus or isthmus - 3 h after last egg posture). Calcium (total and ionic), inorganic phosphorus, alkaline phosphatase, albumen, and total proteins (both used for the calculation of the ionic calcium) in the serum were analyzed with commercial kits (Bioclin Laboratory, Minas Gerais, Brazil). We observed hens and estimated the period after lost egg to determine the phase of next egg formation. One hen per experimental unit was weighed and slaughtered (n = 30) by cervical dislocation. Birds were slaughtered between 11:00 Am and 12:00 Pm (because at this time, eggs were probably located between the infundibulum and magnum) for the collection of the left and right tibiotarsus and intestine samples. Bone

parameters were measured according to the methods described by Zhang and Coon (1997) and Park et al. (2003). After that, bones were dissected, weighed, and frozen. The left tibiotarsus was used for the calculation of the Seedor index (SI) and strength index, and the percentage of ash, calcium (Ca), and phosphorus (P) in the mineral matrix. SI was calculated according to Seedor et al. (1991), while tibiotarsus strength index was obtained according to Monteagudo et al. (1997). After thawing, bone strength analysis was performed in a texturometer TA.XT2i Texture Analyzer with a 29 Warner-Bratzler Shear Force probe (Texture Technologies Corp. and Stable Micro Systems Ltd., Hamilton, MA). The equipment was calibrated with a standard weight of 5 kg at a speed of 3 mm per min. Force was applied to the tibiotarsus diaphysis and bone breaking strength values, in kgf, were divided by bone weight, and expressed in kilogram per gram. The same bones were later defatted in petroleum ether for 24 h, dried in an oven at 65◦ C, ground, again dried in an oven at 105◦ C for 12 h, and finally calcined in a muffle furnace at 600◦ C to obtain ash (AOAC International, 2007). Ash was used to analyze mineral content and to estimate the percentage of Ca and P.

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Ingredient, % Corn (8.8% CP) Soybean meal (45% CP) Dicalcium phosphate Limestone Soybean oil Salt Sodium bicarbonate DL-Methionine (98%) L-Valine (96.5%) L-Lysine HCl (78%) L-Threonine (98%) Vitamin and mineral premix1 L-glutamic acid (98.5%) Kaolin Antioxidant2

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L-GLUTAMIC ACID FOR LAYING HENS

Intestinal Morphology The same laying hens used for bone collection had their intestines collected. All the intestines were isolated, weighed, and measured. Part of the middle portion of the jejunum was collected, opened longitudinally, washed with PBS, and fixed in 10% formaldehyde and phosphate buffer 0.1 M with pH 7.3. The parts were histologically processed, embedded into paraffin, sectioned into 7-μm segments, stained with hematoxylin and eosin, and analyzed under a light microscope with the use of a software for the analysis of digital images (Motic Images Plus 2.0). In each hen (n = 6 per treatment), 30 well-oriented villi and 30 longitudinally sectioned crypts were measured. Villus height was measured from the extremity of the villi to the villus–crypt junction, while width was measured in the middle portion of the villi. Cuts of the jejunum, with approximately 5 μm, were fixed in paraffin, prepared in marked slides, and cell proliferative activity was analyzed with proliferating cell nuclear antigen (PCNA) immunohistochemistry. The paraffin was removed from the cuts by washing them in xylene baths, and dehydrated in a decreasing series of ethanol (99 to 70%) followed by distilled water. Endogenous peroxidase blocking was performed with the hydrogen peroxide block of the EXPOSE mouse and rabbit-specific HRP/DAB detection IHC kit (ab94710) (Abcam, CA bridge, MA, USA) for 10 min. Then, the cuts were submitted to a microwave at 700 MHz in a citrate buffer 0.1 M, pH 6.0 for 15 min. The block of

non-specific proteins was performed via pre incubation with the protein block of the same kit for 10 min in a humid chamber. PCNA (anti-PCNA; FL-261; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was detected with a rabbit polyclonal antibody against the amino acids 1–261 of the human PCNA. The cuts were incubated with the anti-PCNA primary antibody (1:200) overnight in a humid chamber at 4◦ C. Finally, amplification of the primary antibody reaction was performed using an anti-rabbit secondary antibody from the kit for 10 min at ambient temperature, followed by washing in PBS and incubation with the goat antirabbit HRP conjugate from the kit for 15 min. After washing in PBS, the reaction was seen through the 3,3 -diaminobenzidine (DAB) plus substrate + DAB chromogen from the kit for 2 to 3 min. The cuts were washed in running water and counterstained with hematoxylin, after which they were mounted in Permount and dehydrated in increasing concentrations of ethanol and xylene (5 min each). The positive reaction was visualized in the form of a brown stain in the cell nucleus. As a negative control, the primary antibody was omitted from some cuts and replaced by PBS.

Statistical Analysis The data were analyzed using SAS statistical program (SAS, 2009). After the variance analysis, when differences were observed, the degrees of freedom were deployed on polynomials and analyzed by regression for the different linear and quadratic relationships and interaction (P < 0.05). A 5 × 3 factorial arrangement of 5 dietary L-glutamic acid and blood collections at 3 different times was used from 53 to 69 wk. The statistical analyses for the blood serum were performed through the comparison among means of the variables as a function of the blood collection time; this was achieved by using Tukey’s multiple comparisons of means at 5% of significance.

RESULTS AND DISCUSSION Egg production and egg quality of laying hens fed increasing dietary L-glutamic levels between 53 and 69 wk of age are shown in Table 2. In the regression analysis performed among the added levels of Glu, no effect was observed on the productive performance. Concerning egg quality, the percentage of Ca in the shell demonstrated an increasing linear effect (P < 0.05). Mean bone quality data from the tibiotarsus of the laying hens at 69 wk of age are shown in Table 3. The Glu level influenced the SI and tibiotarsal strength index with decreasing and increasing linear responses (P < 0.05), respectively. The SI is an indicator of bone quality that takes bone volume into consideration. These results suggest that the bones of laying hens were probably structurally reinforced as a function of the Glu

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The right tibiotarsus was used to determine the optical densitometry in radiographic images in comparison to an aluminum scale with 10 degrees of 1 mm (penetrometer). Bones were placed under a periapicaltype radiographic film (Kodak Intraoral E-Speed Film, size 2), and radiographed with a dental X-ray machine (DabiAtlante, model Spectro 70X electronic, Ribeir˜ ao Preto, Brazil), with 70-kVp beam energy, 8-μA beam intensity, 0.2 s integration time, and containing a step wedge. The X-ray beam was perpendicularly positioned in relation to the film, with a focus-film distance of 10 cm. Radiographic films were developed in an automatic processor (Revel Industry and Trade of 43 equipment Ltd), during 150 s in a Kodak RP X-Omat solution. Radiographies were digitized with Image Tool (version 3.0) software. Digital images were analyzed in the Adobe Photoshop CS6 histogram. Five areas of each penetrometer degree (1 to 5 mm) were analyzed, and the equation was calculated based on the obtained values. The same procedure was performed for the diaphysis of each bone, and the obtained values were applied in the equation to obtain the respective bone mineral density values expressed in millimeters of aluminum (mm Al). The greater the value in millimeters of aluminum, the greater the radiopacity and, consequently, the higher the bone density.

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Table 2. Productive performance and egg quality mean results from laying hens fed increasing L-glutamic acid supplementation levels between 53 and 69 wk of age. Dietary L-glutamic acid (%) Variable

1

0.2

0.4

0.6

0.8

Means

SEM

P value

82.10 94.92 1.38 1.77 65.31 53.58 1.079 86.51 8.57 0.362 3.48 35.97 84.19

81.72 95.48 1.40 1.79 65.82 53.25 1.078 86.61 8.43 0.354 3.42 35.23 83.32

77.83 94.30 1.47 1.89 65.08 49.58 1.079 86.40 8.48 0.364 3.62 35.93 85.64

81.33 95.61 1.41 1.82 65.34 52.49 1.081 86.79 8.44 0.358 3.47 36.43 86.18

81.40 95.54 1.42 1.82 65.60 52.95 1.079 87.50 8.51 0.363 3.51 36.42 83.23

81.04 95.23 1.42 1.82 65.43 52.27 1.079 86.76 8.49 0.360 3.51 35.98 84.50

0.447 0.357 0.009 0.012 0.139 0.426 0.001 0.286 0.031 0.001 0.036 0.148 0.868

0.517 0.629 0.211 0.165 0.821 0.540 0.323 0.297 0.410 0.494 0.714 0.0471 0.823

ˆ = 35.582 + 1.0221x; r2 = 0.80). Linear effect (Y

Table 3. Tibia bone quality of 69-wk-old laying hens fed increasing L-glutamic acid supplementation levels. Dietary L-glutamic acid, % Variable Relative weight (g) Length (mm) Diameter (mm) Seedor Index (mg/mm) Bone breaking strength (kgf) Bone breaking strength (kg/g) Bone robusticity index Tibia ash (%) Ca in the ash (%) P in the ash (%) Ca:P ratio in the ash OP (mm Al)

0.0

0.2

0.4

0.6

0.8

Mean

SEM

P value

0.50 112.58 6.65 66.50 19.38 2.59 54.80 51.57 27.72 15.34 1.83 2.24

0.48 113.22 6.895 66.89 18.57 2.45 54.85 52.61 27.98 14.49 1.94 2.19

0.46 111.94 6.64 66.33 20.68 2.78 54.96 53.72 27.08 14.10 1.93 2.20

0.48 113.75 6.62 64.71 19.23 2.63 55.64 53.93 27.07 14.44 1.89 2.16

0.46 113.27 6.68 62.21 21.62 2.43 56.36 49.38 26.03 13.20 1.83 2.17

0.47 112.95 6.70 65.03 19.90 2.58 55.45 52.85 27.75 14.82 1.88 2.17

0.474 0.535 0.072 0.699 0.631 0.083 0.222 0.567 0.560 0.411 0.027 0.032

0.874 0.617 0.698 0.0271 0.419 0.817 0.0112 0.436 0.760 0.794 0.823 0.206

OP—Optical densitometry in radiographic images 1 ˆ = 54.5452 + 1.943x; r2 = 0.95). Linear effect (P < 0.05); (Y 2 ˆ = 67.4703 – 5.3641x; r2 = 0.94). Linear effect (P < 0.05); (Y

added to the diet, which is demonstrated by the linear effects in the SI and strength index. Ash, which represents all bone matter, did not signal whether there were differences in relation to cortical and medullar bone fractions. Although no effect was observed in bone breaking strength, the results indicate that the cortical bone remained similar among treatments, while they did not indicate medullar bone condition. Knott and Bailey (1999) reported that medullar bone in hens at 49 wk of age did not participate in bone breaking strength due to the large differences in the organization of the organic matrix (collagens and other proteins) and the distribution of the mineral matrix (Ca and inorganic P). In broilers, studies using L-glutamic acid showed that bone deformation in legs can appear as a result of nonspecific nitrogen deficiency (Moraes et al., 1984, Ribeiro et al. 1995). Changes in non-collagen protein concentrations, responsible for bone fragility, interfere with the complete mineralization and/or normal architecture of bone (Vetter et al., 1991; Young, 2003). Another study regarding Glu supplementation in purified diets of broiler chickens (1 to 14 D) showed that 10% Glu and

15,000 IU of vitamin D3 optimize bone growth, bone organic matter content, and reduce leg problems and non-collagenous protein in femur ant tibiotarsus (Silva et al., 2001a,b). In laying hens with an average of 50-wk production, previous authors observed a reduction in bone strength with considerable cortical bone loss (Whitehead and Fleming, 2000). Cortical bone loss is critical because birds cannot replace it during the laying period (Taylor and Dacke, 1984). The decreased structural and trabecular bone do not cause changes in the external dimensions of large bones, once the reabsorption of cortical bone is confined to the endosteal surface. However, cortical bone thinning and trabecular bone integrity loss result in brittle bones that are more susceptible to fractures (Gregory and Wilkins, 1989). Although differences in relation to shell weight, thickness, and breaking strength were not observed among treatments, the addition of Glu provided increased amounts of Ca in the eggshell. This suggests that there was a metabolic increase in Ca excretion by the uterus and the fixation of this mineral in the eggshell without injuring the skeleton of these animals. This increase

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Egg production (%) Feed intake (g/bird/day) Feed conversion (kg/dz) Feed conversion (kg/kg) Egg weight (g) Egg mass (g) Specific weight (g/cm3 ) Haugh unit Eggshell (%) Thickness (mm) Eggshell shell strength (kg-force) Eggshell Ca (%) Eggshell mineral matter (%)

0.0

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L-GLUTAMIC ACID FOR LAYING HENS Table 4. Blood circulating enzymes and ions from blood collected from 69-wk-old laying hens fed increasing dietary L-glutamic acid levels at 3 different times. Alkaline phosphatase, U/L

Variable

Total calcium, mg/dL

Ionic calcium, mg/dL

Ionic phosphorus, mg/dL

364.65 350.47 344.29 371.95 361.27

14.64 14.66 15.04 14.71 14.96

10.77 10.82 11.06 10.68 11.03

5.94 5.82 6.84 5.73 6.64

Blood collection (time) 04:00 pm 03:00 am 12:00 pm SEM

384.26 358.09 331.04 12.234

15.64a 15.08a 13.68b 0.147

10.93a 11.93a 10.21b 0.115

6.45a 7.18a 4.99b 0.250

P value main effect Glu Time Interaction a,b

0.918 0.999 0.473

0.424 0.0001 0.587

0.637 0.0001 0.285

0.455 0.0009 0.369

Means with different superscript letter differ (P < 0.05) based on Tukey’s significant difference test.

Table 5. Intestinal morphology of 60-wk-old laying hens fed increasing dietary L-glutamic acid levels. Dietary L-glutamic acid, % Variable

0.0

0.2

0.4

0.6

0.8

Mean

SEM

P value

Relative weight (%) Length (cm)

2.74 139.00

3.26 152.0

2.60 151.00

2.28 139.00

2.66 150.00

2.26 132.80

0.063 2.393

0.296 0.923

Jejunun Villus weight (μ m) Villus width (μ m) Crypt depth (μ m) Villus:Crypt ratio

939.39 127.77 152.80 7.23

835.58 130.67 173.89 5.47

837.31 111.12 180.61 4.94

717.18 94.57 144.53 5.23

829.07 112.89 175.58 4.66

680.23 94.86 154.23 4.51

29.176 3.521 3.920 0.213

0.843 0.668 0.076 0.431

may be the result of the greater availability of circulating Ca during shell deposition, associated with greater Ca intestinal absorption and medullar bone deposition, preserving cortical bone. Blood enzymes and circulating ions results in the serum of laying hens at 69 wk of age are shown in Table 4. Glu addition did not influence the levels of Ca, P, and alkaline phosphatase in the serum of hens, and an interaction between the different levels of Glu and blood collection time was not observed. However, collection time presented an isolated effect. Collections were performed at the beginning (04:00 Pm) and end (03:00 Am) of eggshell deposition in the uterus, and during the transit period of the next egg between the infundibulum and magnum (12:00 Pm). Alkaline phosphatase results demonstrated similar mean for each period and were not influenced by the addition of Glu to the diet. A blood collection time effect was expected as a function of eggshell deposition in the uterus of the hens, resulting in physiological fluctuations in Ca and P levels. Ca levels (total and ionic) and P levels differed significantly at the collection at 03:00 Am, as they presented increased serum levels. The increased Ca absorption in the intestine during shell formation is not enough to meet the high Ca requirements. During eggshell calcification in hens, approximately 2 to 2.4 g of Ca is

required to produce the shell for a 60-g egg; however, only 60 to 70% of Ca of the eggshell can be provided via feeding, while the rest should be mobilized from body reserves (Bar, 2009). Moreover, there is Ca mobilization from medullar bone and, after egg laying, osteoblasts are activated for medullar bone regeneration (Whitehead, 2004). Total Ca and P values were similar to those described in previous studies conducted with hens at peak production. Physiologically, P and Ca levels are higher during eggshell calcification than when the uterus is empty; these differences have been described in several studies in laying hens (Clunies et al., 1992; Ghise et al., 2009). The ionic Ca observed in hens represented an average of 73.3% of total Ca. The relative weight and length of small intestine and histomorphology of jejunum results from the laying hens at 69 wk are shown in Table 5. The variables were not influenced by the addition of Glu in the diet. However, crypt depth had a tendency to linear effect (P = 0.076) of treatments. The PCNA immunohistochemistry analysis revealed that the addition of Glu interfered with the quality of the jejunal villi. The observations conducted under a light microscope evidenced that in all treatments there was proliferative activity in the crypts and at the base

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Dietary L-glutamic acid, % 0.0 0.2 0.4 0.6 0.8

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of jejunal villi of hens at 69 wk (Figure 1). In birds fed diets with 2.88 and 3.08% of Glu, positive nuclei were observed in approximately one-third of the villus from the base. However, diets formulated with 3.28 and 3.48% of Glu resulted in an increased proportion of the intestinal villi with nuclei marked positively for PCNA, especially positive nuclei at the villi extremity (Figures 1 and 2). In the negative control slides, where the primary antibody was omitted in the corresponding step, the nuclei did not present reactions demonstrating the specificity of the used antibody (Figure 1F). Glutamine and glutamic acid can be converted into one another in different organs such as the intestines, liver, and kidneys (Wu, 2009), and both are related to intestinal tract development. Glutamine increases intestinal cell proliferation, improving its morphology. The effects of Gln and its metabolites on the development and maintenance of the intestinal mucosa have been described in several animal species. Furthermore,

Gln decreases the expression of the gene involved in the oxidative stress and immune activation, contributing to the maintenance of intestinal mucosal growth (Wang et al., 2008). Broilers that were fed 1% L-glutamine acid since the first day of age presented increased villus height of the duodenum and ileum, and the crypt depth of the duodenum was increased in birds that received high amounts this amino acid. At 14 D, the same effect was observed in the crypt depth of the jejunum (Maiorka et al., 2000). The development of the intestinal mucosa is the result of cell renovation provided by proliferation and differentiation of crypt and villus (Uni et al., 1998). According to Reeds et al. (2000), Glu is the most important substrate for the maintenance of intestinal health and is the main substrate for the oxidative reactions on the mucosa. It also has an important role in the biosynthesis of essential amino acids (proline and arginine), and it is a key factor in mucosal protection by glutathione.

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Figure 1. Jejunum of 69-wk-old laying hens fed increasing dietary L-glutamic acid levels. (A–E) Note positive reaction (brown) for PCNA in the nuclei of crypt cells (cr) for all treatments and along the intestinal villus (vi). More positive cells are found towards the end of the villus as acid L-glutamic acid is added to the feed. (F) Negative control (neg) in which the omission of the primary antibody showed no immunoreactivity. (A) 2.68% Glu; (B) 2.88% Glu; (C) 3.08% Glu; (D) 3.28% Glu; (E) 3.48% Glu. Counterstained by hematoxylin. Scale bar = 100 μ m.

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A study by Uni et al. (1998) showed that stem cells from the crypts in the small intestine of mammals differ mainly with regard to the enterocytes that migrate to the villus until they are expelled from the intestine into the villus extremity. During this migration, the cells acquire functions. In broilers, differently from what occurs in mammals, enterocytes present a proliferative activity that occurs not only in the crypt. However, the exact place where differentiation occurs has not been precisely identified yet. Increased crypt depth was associated to the largest proportion of cells reactive to PCNA in treatments with the addition of Glu in the diet of hens. This is evident that there was increased proliferative activity of the jejunum of the birds in the present study, thereby indicating increased mucosal regenerative ability. Although hens managed in cages do not suffer bacterial challenges such as those that occurs in broilers managed in poultry litter, and nutrient absorptive ability is not required as a function of the fast growth and weight gain of broilers, hens require the maintenance of intestinal health for nutrient absorption. In another study, Uni et al. (2001) demonstrated that cell proliferation in broilers occurs in the crypt and in the villus, with 50% of proliferative cells found in the crypt, 32% in the middle of the villus, and 8% in the apical region of the villus. Geyra et al. (2001) identified in 1-day-old broilers that almost all cells from the 3 intestinal segments were in the proliferation stage in the crypt and in the villus. However, with increased age, the percentage of positive PCNA cells decreased, with approximately 50% of positive PCNA cells in the

crypt and 10 to 40% in the villus 3 D after outbreak. The aforementioned studies were performed in broiler chicks at only a few days of age. However, in adult hens no references have been found for comparison. The intense nucleus proliferation marked by the immunohistochemistry test suggests that Glu acts on the cellular renovation of jejunum cells of adult hens. In conclusion, laying hens fed diets formulated with increased levels of L-glutamic acid between 53 and 69 wk of age presented higher Ca eggshell deposition. The addition of glutamic acid also influenced the tibiotarsus SI and strength index and had a qualitative increment in cellular proliferation of the jejunum mucosa. Among the experimental Glu levels, the addition of 0.8% L-glutamic acid resulted in improved bone parameters in 69-wk-old laying hens and, consequently, helped to improve quality and duration of the productive period.

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Figure 2. Jejunum of 69-wk-old laying hens fed increasing dietary L-glutamic acid levels. (A) Note sectional crypt (cr) of the jejunum with PCNA-labeled nuclei, viewable brown (thick arrow) and continuation of the crypt to the surface of the villi (vi) where PCNA cell proliferation can be observed and some negative cells are displayed in blue (thin arrow). (B–D) Extremity of the jejunum villi in detail. In hens with low inclusion of Glu in the diet (B), the extremity of villus of the jejunum has negative enterocytes (en) nuclei to PCNA (thin arrow) while in the treatments with greater inclusion of Glu (C and D) many cells present positive nuclei PCNA-labeled (thick arrow) even at the extremity of the villi. (A, D) 3.48% Glu; (B) 2.88% Glu; (C) 3.28% Glu. Counterstained by hematoxylin. Scale bar = 30 μ m.

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