Circadian regulation of calcium and phosphorus homeostasis during the oviposition cycle in laying hens.

Maintenance of calcium and phosphorus homeostasis in laying hens is crucial for preservation of skeletal integrity and eggshell quality, though physiological regulation of these systems is incompletely defined. To investigate changes in mineral and vitamin D3 homeostasis during the 24-h egg formation cycle, 32-wk-old commercial laying hens were sampled at 1, 3, 4, 6, 7, 8, 12, 15, 18, 21, 23, and 24 h post-oviposition (HPOP; n ≥ 4). Ovum location and egg calcification stage were recorded, and blood chemistry, plasma vitamin D3 metabolites, circulating parathyroid hormone (PTH), and expression of genes mediating uptake and utilization of calcium and phosphorus were evaluated. Elevated levels of renal 25-hydroxylase from 12 to 23 HPOP suggest this tissue might play a role in vitamin D3 25-hydroxylation during eggshell calcification. In shell gland, retinoid-x-receptor gamma upregulation between 6 and 8 HPOP followed by subsequently increased vitamin D receptor indicate that vitamin D3 signaling is important for eggshell calcification. Increased expression of PTH, calcitonin, and fibroblast growth factor 23 (FGF23) receptors in the shell gland between 18 and 24 HPOP suggest elevated sensitivity to these hormones toward the end of eggshell calcification. Shell gland sodium-calcium exchanger 1 was upregulated between 4 and 7 HPOP and plasma membrane calcium ATPase 1 increased throughout eggshell calcification, suggesting the primary calcium transporter may differ according to eggshell calcification stage. Expression in shell gland further indicated that bicarbonate synthesis precedes transport, where genes peaked at 6 to 7 and 12 to 18 HPOP, respectively. Inorganic phosphorus transporter 1 (PiT-1) expression peaked in kidney between 12 and 15 HPOP, likely to excrete excess circulating phosphorus, and in shell gland between 18 and 21 HPOP. Upregulation of FGF23 receptors and PiT-1 during late eggshell calcification suggest shell gland phosphorus uptake is important at this time. Together, these findings identified potentially novel hormonal pathways involved in calcium and phosphorus homeostasis along with associated circadian patterns in gene expression that can be used to devise strategies aimed at improving eggshell and skeletal strength in laying hens.

Effect of phytase and limestone particle size on mineral digestibility, performance, eggshell quality, and bone mineralization in laying hens.

The effect of microbial phytase and limestone particle size (LmPS) was assessed in Lohmann Tradition laying hens from 31 to 35 wk of age. Seventy-two hens were used in a completely randomized trial according to a 2 × 2 factorial arrangement with 2 levels of phytase/basal available P (aP); 0 FTU/kg with 0.30% aP or 300 FTU/kg with 0.15% aP, and 2 limestone particle sizes; fine particles (FL, <0.5 mm) or a mix (MIX) of 75% coarse limestone (CL, 2-4 mm) and 25% FL. Diets contained equivalent levels of Ca (3.5%), phytic P (PP; 0.18%), and aP (0.30%) considering the P equivalency of phytase. Thus, dietary treatments were FL0 and MIX0 without phytase, and FL300 and MIX300 with 300 FTU/kg phytase. Performance were recorded daily and eggshell quality (eggshell weight proportion, weight, thickness, and breaking strength) was measured weekly. At the end of the trial, bone parameters (tibia breaking strength, elasticity, and ash) and the apparent precaecal digestibility (APCD) of P and Ca were determined. No differences were observed between treatments in feed intake, FCR and bone parameters. Addition of MIX increased the eggshell proportion, weight and thickness in groups receiving no phytase (+6.5, +6.9, and +4.5%, respectively) while no effect was observed in groups receiving phytase (Phytase × LmPS, P < 0.05). In hens receiving FL, the APCD of P was lower in diets supplemented with phytase (-14 percentage points; Phytase × LmPS, P < 0.001). A higher phytate disappearance was observed in hens fed diets with phytase in combination with MIX (Phytase × LmPS, P = 0.005). Phytase and MIX together increased the APCD of Ca by 7.3 percentage points (Phytase × LmPS, P < 0.001). In conclusion, addition of CL could limit the formation of Ca-phytate complex thus improving the response of the birds to phytase compared to FL.

Eggshell Membrane Ameliorates Hyperuricemia by Increasing Urate Excretion in Potassium Oxonate-Injected Rats.

Hyperuricemia is the primary cause of gouty arthritis and other metabolic disorders. Eggshell membrane (EM) is an effective and safe supplement for curing pain and stiffness connected with osteoarthritis. However, the effect of EM on hyperuricemia is unclear. This study determines the effects of EM on potassium oxonate-injected hyperuricemia. Uric acid, creatinine, blood urea nitrogen concentrations in the serum, and xanthine oxidase activity in the liver are measured. Protein levels of renal urate transporter 1 (URAT1), organic anion transporters 1 (OAT1), glucose transporter 9 (GLUT9), and ATP-binding cassette transporter G2 (ABCG2) in the kidney are determined with renal histopathology. The results demonstrate that EM reduces serum uric acid levels and increases urine uric acid levels in hyperuricemic rats. Moreover, EM downregulates renal URAT1 protein expression, upregulates OAT1 and ABCG2, but does not change GLUT9 expression. Additionally, EM does not change xanthine oxidase activity in the liver or the serum. EM also decreases uric acid uptake into oocytes expressing hURAT1. Finally, EM markedly reduces renal inflammation and serum interleukin-1ß levels. These findings suggest that EM exhibits antihyperuricemic effects by promoting renal urate excretion and regulating renal urate transporters. Therefore, EM may be useful in the prevention and treatment of gout and hyperuricemia.

Effects of phytase supplementation on eggshell and bone quality, and phosphorus and calcium digestibility in laying hens from 25 to 37 wk of age.

Effects of dietary available phosphorus (aP) and Ca levels and an Escherichia coli 6-phytase supplementation were studied in Lohmann LSL-Lite hens from 25 to 37 wk of age. Eighty-four hens were used in a completely randomized design with 7 treatments. The treatments were a positive control (PC) diet with 0.45% aP, 3.70% Ca, and 0.16% Na from 25 to 28 wk and 0.38% aP, 3.73% Ca, and 0.15% Na from 29 to 37 wk; a negative control (NC) diet, similar to the PC diet, with 0.22% aP, 3.00% Ca, and 0.13% Na from 25 to 28 wk and 0.19% aP, 3.02% Ca, and 0.13% Na from 29 to 37 wk; the NC diets supplemented with phytase at 150 (NC + 150), 300 (NC + 300), 600 (NC + 600), or 1,200 (NC + 1,200) phytase unit (FTU)/kg; and the PC diet supplemented with phytase at 1,200 (PC + 1,200) FTU/kg. Hen performance, eggshell, and bone quality were measured on a 4-wk basis. Bone breaking strength and ash and apparent ileal digestibility (AID) of P and Ca were determined at 37 wk. One- and 2-way ANOVA were conducted, and Tukey's range test was used to compare multiple means where P ≤ 0.05. No differences in hen performance, eggshell quality, bone breaking strength, bone ash, and P digestibility were observed between the PC and the NC treatments. The NC hens had lower cortical (P < 0.001) and trabecular + medullary bone mineral density (P = 0.004) and total bone mineral content (P < 0.001) than the PC hens. The PC + 1,200 increased cortical bone mineral density (P < 0.001). The reductions of aP and Ca in the NC diet were not deficient for performance but had a minor impact on bone mineralization. The NC + 600 and NC + 1,200 increased AID of P (P = 0.024), and all phytase treatments except the NC + 150 increased AID of Ca (P = 0.010) compared with the NC diet.

Effect of high concentrations of dietary vitamin D3 on pullet and laying hen performance, skeleton health, eggshell quality, and yolk vitamin D3 content when fed to W36 laying hens from day of hatch until 68 wk of age.

The objective of this experiment was to investigate the effects of various dietary concentrations of vitamin D3 (D3) on pullet and laying hen performance, eggshell quality, bone health, and yolk D3 content from day of hatch until 68 wk of age. Initially, 440 Hy-line W36-day-old chicks were randomly assigned to 5 dietary treatments: 1,681 (control); 8,348; 18,348; 35,014; 68,348 IU D3/kg. At 17 wk of age, pullets were assigned to experimental diets with 12 replicate groups of 6 birds. At 17 wk of age, pullets fed diets containing 8,348 and 35,014 IU D3/kg had an increased bone mineral density in comparison to the control fed birds (P ≤ 0.01). Body weights of pullets fed the diet with 68,348 IU D3/kg were lower than other treatments (P ≤ 0.01). Hen-housed egg production (HHEP) of hens fed the 35,014 IU D3/kg diet was increased in comparison to control-fed hens (P ≤ 0.01), whereas HHEP of those fed 68,348 IU D3/kg diet was reduced in comparison to all other treatments (P ≤ 0.01). Shell breaking strength of eggs from hens fed 8,348, 35,014 and 68,348 IU D3/kg was increased in comparison to eggs from control-fed birds (P ≤ 0.01). Fat-free tibia ash content of hens fed any of the diets supplemented with D3 (8,348 to 68,348 IU D3/kg) was increased in comparison to control-fed hens (P ≤ 0.05). Yolk D3 content increased linearly with dietary D3 and the D3 transfer efficiency for the control, 8,348 IU, 18,348 IU, 35,014 IU, and 68,348 IU D3 treatments were 8.24, 10.29, 11.27, 12.42, and 12.06%, respectively. These data suggest that supplementation of dietary D3 up to 35,014 IU D3/kg feed maintained if not increased laying hen performance and enhanced pullet and laying hen skeletal quality as well as yolk D3 content and eggshell quality. Feeding pullets at a higher level 68,348 IU of D3 resulted in reduced growth and ultimately decreased performance of laying hens.

Estimation of calcium requirements for optimal productive and reproductive performance, eggshell and tibial quality in egg-type duck breeders.

Optimizing the dietary calcium (Ca) level is essential to maximize the eggshell quality, egg production and bone formation in poultry. This study aimed to establish the Ca requirements of egg-type duck breeders from 23 to 57 weeks of age on egg production, eggshell, incubation, tibial, plasma and ovary-related indices, as well as the expression of matrix protein-related genes. Totally, 450 Longyan duck breeders aged 21 weeks of age were allotted randomly into five treatments, each with six replicates of 15 individually caged birds. The data collection started from 23 weeks of age and continued over the following 35 weeks. The five groups corresponded to five dietary treatments containing either 2.8%, 3.2%, 3.6%, 4.0% or 4.4% Ca. The tested dietary Ca levels increased (linear, P <0.01) egg production and egg mass, and linearly improved (P <0.01) the feed conversion ratio (FCR). Increasing the dietary Ca levels from 2.8% to 4.4% increased (P <0.01) the eggshell thickness and eggshell content. The tested Ca levels showed a quadratic effect on eggshell thickness and ovarian weight (P <0.01); the highest values were obtained with the Ca levels 4.0% and 3.6%, respectively. Dietary Ca levels affected the small yellow follicles (SYF) number and SYF weight/ovarian weight, and the linear response (P <0.01) was significant vis-à-vis SYF number. In addition, dietary Ca levels increased (P <0.05) the tibial dry weight, breaking strength, mineral density and ash content. Plasma and tibial phosphorus concentration exhibited a quadratic (P <0.01) response to dietary Ca levels. Plasma calcitonin concentration linearly (P <0.01) increased as dietary Ca levels increased. The relative expression of carbonic anhydrase 2 in the uterus rose (P <0.01) with the increment of dietary Ca levels, and the highest value was obtained with 3.2% Ca. In conclusion, Longyan duck breeders fed a diet with 4.0% Ca had superior eggshell and tibial quality, while those fed a diet with 3.6% Ca had the heaviest ovarian weights. The regression model indicated that the dietary Ca levels 3.86%, 3.48% and 4.00% are optimal levels to obtain maximum eggshell thickness, ovarian weight and tibial mineral density, respectively.

Long-term effects of Buttiauxella sp. phytase on performance, eggshell quality, apparent ileal Ca and P digestibility, and bone properties of white egg layers.

Adequate dietary Ca and available phosphorus (avP) are essential to long-term egg production and bone health in laying hens. The effects of dietary Ca and avP levels and Buttiauxella sp. phytase (BSP) were studied in Lohmann LSL Lite hens from 30 to 70 wk of age (woa). Hens (n = 456; 4 per cage) were fed either a primary breeder recommendation-based diet (positive control; PC); the PC with avP and Ca levels reduced by 0.146 and 0.134% of the diet, respectively, without (NC) or with 300 FTU/kg BSP (NC+BSP). Egg production, BW, feed intake, FCR, and eggshell quality from 30 to 70 woa, and apparent ileal digestibility of P (AIDP) and Ca (AIDCa), and bone quality at 32, 48, and 70 woa were measured. The avP and Ca levels in the NC diet were not clinically deficient, as most parameters were unaffected by diet. Hen BW from 34 to 70 woa tended to be 2.9% greater (P = 0.076) for PC and NC+BSP compared to NC. Mid-diaphysis cortical bone mineral content (CBMC) tended to be 10% and 9% higher (P = 0.065) in the NC+BSP hens than in NC hens at 48 and 70 woa, respectively. AIDP of NC+BSP was 24% greater (P = 0.034) than of NC at 32 woa and tended to be 18% greater (P = 0.082) than AIDP of PC at 48 woa, and 25% lower than of NC and PC at 70 woa (P = 0.028). AIDCa was 25% lower for NC+BSP than PC at 48 woa only (P = 0.037). The avP and Ca sufficiency in the NC diet limited the opportunity to determine a phytase effect. Although the supplemental BSP tended to increase BW and 48 and 70 woa CBMC, and increased 32 woa AIDP, the efficacy of BSP could not be determined due to the lack of an NC effect on most parameters. Commercial laying hens can maintain health and productivity at lower than recommended levels of dietary Ca and avP; phytase supplementation may allow for even further reductions.

The effect of different dietary levels of hybrid rye and xylanase addition on the performance and egg quality in laying hens.

1. In this study, 240 ISA Brown hens were fed diets containing different levels of hybrid rye, and the influence of xylanase addition on laying performance and egg quality was evaluated. 2. Birds were allocated to 10 treatment groups with 12 replicates (cages) of two hens and were fed, from week 26 to 50, isocaloric and isonitrogenous experimental diets. A 5 × 2 experimental arrangement was applied, using diets with increasing level of rye (0%, 10%, 15%, 20% or 25%) with or without xylanase supplementation (200 mg/kg of feed; Ronozyme WX (CT) with minimum xylanase activity of 1,000 FXU/g). 3. Increasing dietary level of rye did not affect daily mass of eggs, mean egg weight or feed conversion ratio (P > 0.05). Laying rate decreased in all groups fed with rye. Egg and eggshell quality indices were unaffected by dietary rye grain (P > 0.05); however, rye inclusion significantly decreased yolk colour on the DSM scale (P < 0.05). In comparison with the control group, high dietary levels of rye (25%) significantly increased viscosity of small intestine content (P < 0.05). Diet supplementation with xylanase had no significant effect on egg production indices and egg quality (except for yolk colour) but decreased the viscosity of intestinal content in laying hens fed high levels of rye (P < 0.05). 4. The results of this experiment suggest that rye may be incorporated to a level of 25% in the diet of laying hens without any strong negative effect on egg performance, while xylanase added to high-rye grain reduced the viscosity of intestinal content; however, it did not positively affect the laying performance or egg quality.

Epigallocatechin-3-gallate protected vanadium-induced eggshell depigmentation via P38MAPK-Nrf2/HO-1 signaling pathway in laying hens.

It has been demonstrated that tea polyphenol (TP) epigallocatechin-3-gallate (EGCG) can confer protection against vanadium (V) toxicity in laying hens; however, our understanding of the molecular mechanisms beyond this effect are still limited. In this study, 360 hens were randomly assigned to the 3 groups to study whether the potential mechanism P38MAPK-Nrf2/HO-1 signaling pathway is involved in the protective effect of EGCG on eggshell pigmentation in vanadium challenged laying hens. Treatments included a control group, a 10 mg/kg V (V10), and a V10 plus 130 mg/kg of EGCG group (V10+EGCG130). Both eggshell color and protoporphyrin IX were decreased in the V10 group compared with the control diet, while EGCG130 treatment partially improved shell color and protoporphyrin IX (P < 0.05). The V10 exposure induced higher cell apoptosis rate and oxidative stress in birds as evidenced by the histological apoptosis status, decreased uterine glutathione-S transferase (GST) and high abundance of malondialdehyde (MDA) compared with the control group, whereas EGCG130 markedly alleviated oxidative stress via reducing MDA generation (P < 0.05). Dietary vanadium reduced ferrochelatase, NF-E2-related factor 2 (Nrf2), and heme oxygenase (HO-1) mRNA expression, while EGCG up-regulated Nrf2 and HO-1 expression (P < 0.05). Protein levels of Nrf2, HO-1 and phospho-p38 (P-P38) MAPK were reduced in V10 group, while dietary supplementation with 130 mg/kg EGCG markedly increased Nrf2, HO-1 and P-P38 MAPK protein levels in the uterus compared with the V10 group (P < 0.01). In conclusion, EGCG improved eggshell color and antioxidant system in V10-challenged hens, which seems to be associated with P38MAPK-Nrf2/HO-1 signaling pathway.

Copper requirements of broiler breeder hens.

One-hundred-twenty Cobb 500 hens, 20 wk of age, were randomly allocated into individual cages with the objective of estimating Cu requirements. After being fed a Cu deficient diet for 4 wk, hens were fed diets with graded increments of supplemental Cu (0.0; 3.5; 7.0; 10.5; 14; and 17.5 ppm) from Cu sulfate (CuSO4 5H2O), totaling 2.67; 5.82; 9.38; 12.92; 16.83; and 20.19 ppm analyzed Cu in feeds for 20 weeks. Estimations of Cu requirements were done using exponential asymptotic (EA), broken line quadratic (BLQ), and quadratic polynomial (QP) models. Obtained Cu requirements for hen d egg production and total settable eggs per hen were 6.2, 7.3, and 12.9 ppm and 8.1, 9.0, and 13.4 ppm, respectively, using EA, BLQ, and QP models. The QP model was the only one having a fit for total eggs per hen with 13.1 ppm Cu as a requirement. Hemoglobin, hematocrit, and serum Cu from hens had requirements estimated as 13.9, 11.3, and 18.5, ppm; 14.6, 13.0, and 19.0 ppm; and 16.2, 14.6, and 14.2 ppm, respectively, for EA, BLQ, and QP models. Hatching chick hemoglobin was not affected by dietary Cu, whereas requirements estimated for hatching chick hematocrit and body weight and length were 10.2, 12.3, and 13.3 ppm using EA, BLQ, and QP models; and 6.8 and 7.1 ppm, and 12.9 and 13.9 ppm Cu using EA and BLQ models, respectively. Maximum responses for egg weight, yolk Cu content, and eggshell membrane thickness were 14.9, 12.7, and 15.1 ppm; 15.0, 16.3, and 15.7 ppm; and 7.3, 7.8, and 14.0 ppm Cu, respectively, for EA, BLQ, and QP models. Yolk and albumen percentage were adjusted only with the QP model and had requirements estimated at 11.0 ppm and 11.3 ppm, respectively, whereas eggshell mammillary layer was maximized with 10.6, 10.1, and 14.4 ppm Cu using EA, BLQ, and QP models, respectively. The average of all Cu requirement estimates obtained in the present study was 12.5 ppm Cu.

Calcium particle size effects on plasma, excreta, and urinary Ca and P changes in broiler breeder hens.

An experiment was conducted using non-colostomized and colostomized broiler breeder hens to determine the effects of feeding limestone of 2 different mean particle sizes (185 microns and 3490 microns) on P excretion, total P and Ca retention, and urinary P and Ca excretion during a 6-week feeding study. Additionally, changes in plasma inorganic P (iP) and ionic Ca (Ca++) and urinary excretion of P and Ca were determined in one egg laying cycle of 24 hours. One-hundred-fifty non-colostomized and 6 colostomized broiler breeder hens, 30 wk of age, were divided into 2 groups and fed broiler breeder diets supplemented with either small particle or large particle limestone. Two % acid insoluble ash (Celite) was added to the feed as a marker. Diets, excreta, and urine samples were analyzed for total P and Ca by ionic coupling plasma (ICP) analysis. The non-colostomized breeders fed large particle limestone compared to small limestone particles produced a significant increase in percent tibia ash (P < 0.0001) and egg specific gravity (P = 0.0382), but P excretion approached a tendency of being reduced (P = 0.1585). The urinary total P and Ca (∼18 and 9%, respectively) of total P and Ca excretion for breeders fed both sizes of limestone was not significantly different in the colostomized breeders. In plasma, both iP and Ca++ reached a peak during 18 to 20 h and 20 to 24 h post oviposition for smaller and larger particle sized limestone fed groups, respectively. The maximal excretion of urinary P was found during 11 to 20 h post oviposition, whereas urinary Ca peaked during 0 to 11 h post oviposition for both smaller and larger particle sized limestone supplemented groups. In summary, the findings indicate that the particle size (smaller and larger) of calcium source did not significantly influence the quantitative total urinary excretion of Ca and P but did influence the timing of Ca and P excretion.

Effects of carbohydrase enzyme supplementation on performance, eggshell quality, and bone parameters of laying hens fed on maize- and wheat-based diets.

1. This study was conducted to determine the effects of enzyme supplementation of maize/wheat-based diets on the performance, egg quality, and serum and bone parameters of laying hens. 2. During the 12-week experimental period, a total of 72 laying hens aged 52 weeks were randomly distributed among 6 experimental groups. Each experimental group contained 4 replicates, each with three birds. The experiment was a randomised design consisting of a 3 × 2 factorial arrangement, with three levels of wheat substitution and two levels of enzyme (xylanase: 1500.00 U/kg, ß-glucanase: 100 000 U/kg, cellulase: 1 000 000 U/kg, α-amylase: 160 000 U/kg) inclusion in the diet. Wheat replaced 0, 50, or 100% of maize with or without 1.0 g/kg enzyme supplementation in iso-nitrogenous and iso-caloric experimental diets. 3. Body weight, egg production, egg weight, egg mass, eggshell thickness, and the feed conversion ratio were adversely affected by the wheat-based diet. The eggshell quality parameters decreased with enzyme supplementation to the diet. 4. Wheat-based diets adversely affected calcium and phosphorus concentrations in the tibia, but the addition of the enzymes to the wheat-based diet prevented the negative effects of wheat-based diets on tibia mineralisation in laying hens. The wheat-based diets tended to reduce plasma mineral contents, and the addition of enzymes tended to affect plasma minerals and biomechanical properties of the tibia positively in laying hens. 5. These results indicate that wheat-based diets in aged laying hens adversely affected the mineral metabolism compared with maize-based diets, and the negative effects of wheat on bone mineralisation can be prevented by enzyme supplementation to the diets in laying hens.

Effects of dietary Mn-methionine supplementation on the egg quality of laying hens.

This study was conducted to investigate the effects of dietary manganese-methionine (Mn-Met) supplementation on the egg quality of laying hens. A total of 480 Jinghong-1 strain layers aged 53 wk were divided into 5 groups with 6 replicates of 16 layers. Birds in the control group were fed a diet supplemented with 60 mg Mn/kg in the form of MnSO4; the birds in other 4 experimental groups were fed a diet supplemented with 20, 40, 60, and 80 mg Mn/kg as Mn-Met, respectively. Dietary Mn-Met treatments significantly affected (P < 0.05) the albumen height, yolk color, and Haugh unit compared to those of the control diet. The Mn contents in the eggshell increased (P < 0.01) significantly by increasing the Mn-Met supplementation, whereas Mn content in eggshell was triple that in the yolk or albumen. Compared with the 60 mg/kg Mn-Met group, the transverse surface in the control group had (P < 0.01) a greater width of mammillary cones, and there were obvious cracks on the outer surface in the control. There was no difference (P > 0.05) in the eggshell gland (ESG) in the expression of calbindin-D28k (CaBP-D28k) mRNA in response to any diet treatment. In conclusion, dietary Mn-Met supplementation increased internal egg quality and the ultrastructure of the eggshell. Compared to the control, 60 mg/kg Mn-Met treatment resulted in improving egg quality, and 20 mg/kg Mn-Met treatment had similar effects the control treatment had on the egg quality. This indicates that the inorganic Mn can be replaced by the lower concentration of Mn-Met.

Dietary manganese supplementation modulated mechanical and ultrastructural changes during eggshell formation in laying hens.

This study evaluated the mechanical and ultrastructural changes during eggshell formation in laying hens by using the optimal levels of organic and inorganic manganese (Mn). A total of 270 62-wk-old Hy-Line Brown laying hens were fed a basal diet containing 25.1 mg Mn per kg feed for 2 wks, after which they were randomly allocated into 3 groups and fed the basal diet (control) or the basal diet supplemented with 120 mg Mn per kg feed from monohydrate Mn sulfate (an inorganic source of Mn), or 80 mg Mn per kg feed from an amino acid-Mn complex (an organic source of Mn) for 12 wks. For each group, 6 replicates of 15 hens were used with one hen per cage. Dietary Mn supplementation significantly increased eggshell-breaking strength and thickness in laying hens (P < 0.05). In neither was the elasticity of their eggshell membranes, measured during the nucleation and mammillary knob formation stages, affected by dietary Mn compared with the control (P > 0.05), whereas the breaking strength of the eggshells was greater at the linear and terminate deposition stages compared with the control (P < 0.05). Dietary Mn supplementation decreased the width of the mammillary knobs and the proportion of mammillary thickness, and increased the proportion of effective thickness of the whole eggshells (P < 0.05). Ultrastructural changes during the eggshell formation indicated that dietary Mn supplementation increased the nucleation site and mammillary knob densities, decreased the mammillary thickness, and increased the proportion of effective thickness and total thickness of the eggshells compared with the control (P < 0.05). Therefore, dietary Mn supplementation can improve the breaking strength and ultrastructure of the eggshells during their formation, and the mammillary and palisade layers are both crucial structures affected by Mn.

Effect of dietary supplementation of organic or inorganic zinc on carbonic anhydrase activity in eggshell formation and quality of aged laying hens.

This study evaluated the effects of different dietary levels and sources of zinc (Zn) on performance and carbonic anhydrase (CA) activity in eggshell formation and quality in aged laying hens. A total of 504 Hy-line Grey layers aged 59 wk were fed a basal diet (Zn, 28.4 mg/kg) for 4 wks, then randomly allocated to 7 groups that were fed a basal diet or a basal diet supplemented with inorganic (ZnSO4·H2O) or organic (amino acid metals, 9.58%) Zn at 35, 70, or 140 mg Zn per kg of feed for 6 weeks. Each group had 6 replicates of 12 hens. Results showed that egg weight decreased linearly with the supplemental level of organic Zn (P < 0.05). Dietary Zn supplementation had linear and quadratic effects on the CA activity in plasma (P < 0.05), and it was higher in the organic Zn-added groups at wks 2 and 4 (P < 0.05). Dietary Zn supplementation had a quadratic effect on the CA activity in the eggshell gland (P < 0.05). Shell thickness was greater in the organic Zn-added groups (P < 0.05), and its relationship with the supplemental level of Zn showed linearly and quadratically, increasing with the organic Zn and with the inorganic Zn at wk 4, while linearly increasing with the inorganic Zn at wk 6 (P < 0.05). At wk 4, the supplemental level of inorganic Zn had a linear effect on shell weight, and linear and quadratic effects on shell index and ratio (P < 0.05), while shell weight, the index, and ratio increased linearly and quadratically with the organic Zn level in the diet (P < 0.05), with more obvious effects in the organic Zn-added groups (P < 0.05). Overall, dietary Zn supplementation, up to 140 mg/kg feed, could increase eggshell thickness by enhancing CA activity in the plasma and eggshell gland of aged layers; thicker eggshells were found in the organic Zn-added groups, but the breaking strength did not increase despite the eggshell thickness increasing.

Effect of dietary supplementation of organic or inorganic manganese on eggshell quality, ultrastructure, and components in laying hens.

This study evaluated the effects of dietary supplemental levels and sources of manganese (Mn) on performance, eggshell quality, ultrastructure, and components in laying hens. A total of 1,080 46-wk-old Jing Brown hens were fed a basal diet (Mn, 32.7 mg/kg) for 2 wks laying and then randomly allocated to 9 groups that were fed a basal diet (control) or the basal diet supplemented with inorganic (MnSO4·H2O) or organic (amino-acid-Mn, 8.78%) Mn at 40, 80, 120, or 160 mg per kg of feed for 8 wks. Each group had 8 replicates of 15 hens. The results showed that dietary Mn supplementation did not affect the performance of hens (P > 0.05). Dietary Mn supplementation resulted in linear and quadratic increases of breaking strength and thickness in both inorganic and organic forms (P < 0.05), but fracture toughness increased quadratically only in organic groups (P < 0.05). Linear and quadratic effects on effective and mammillary thickness were observed with Mn supplementation from inorganic and organic sources (P < 0.05), and lower mammillary thickness was observed in organic groups (P < 0.05). However, the width of mammillary knobs decreased quadratically only with the supplementation of organic Mn (P < 0.05). Dietary Mn supplementation had a quadratic effect on the shell Mn content in both inorganic and organic forms (P < 0.05). Linear and quadratic effects on the content of sulfated glycosaminoglycans (GAGs) were observed only in calcified eggshell with inorganic Mn supplementation (P < 0.05), while the supplementation of organic Mn had a quadratic effect on sulfated GAGs content in both calcified eggshell and membranes (P < 0.05). Overall, dietary Mn supplementation, regardless of the source, could increase breaking strength and thickness by improving the ultrastructure, which partly results from increased sulfated GAGs content in the eggshell. Moreover, the supplementation of organic Mn could increase fracture toughness by decreasing the width of mammillary knobs, which is partially due to increased sulfated GAGs content in the membranes.

Analysis of gene expression and regulation implicates C2H9orf152 has an important role in calcium metabolism and chicken reproduction.

The reproductive system of a female bird is responsible for egg production. The genes highly expressed in oviduct are potentially important. From RNA-seq analysis, C2H9orf152 (an orthologous gene of human C9orf152) was identified as highly expressed in chicken uterus. To infer its function, we obtained and characterized its complete cDNA sequence, determined its spatiotemporal expression, and probed its transcription factor(s) through pharmaceutical approach. Data showed that the complete cDNA sequence was 1468bp long with a 789bp of open reading frame. Compared to other tested tissues, this gene was highly expressed in the oviduct and liver tissues, especially uterus. Its expression in uterus was gradually increased during developmental and reproductive periods, which verified its involvement in the growth and maturity of reproductive system. In contrast, its expression was not significant different between active and quiescent uterus, suggesting the role of C2H9orf152 in reproduction is likely due to its long-term effect. Moreover, based on its 5'-flanking sequence, Foxd3 and Hnf4a were predicted as transcription factors of C2H9orf152. Using berberine or retinoic acid (which can regulate the activities of Hnf4a and Foxd3, respectively), we demonstrated suppression of C2H9orf152 by the chemicals in chicken primary hepatocytes. As retinoic acid regulates calcium metabolism, and Hnf4a is a key nuclear factor to liver, these findings suggest that C2H9orf152 is involved in liver function and calcium metabolism of reproductive system. In conclusion, C2H9orf152 may have a long-term effect on chicken reproductive system by regulating calcium metabolism, suggesting this gene has an important implication in the improvement of egg production and eggshell quality.

Periodical low eggshell temperatures during incubation and post hatch dietary arginine supplementation: Effects on performance and cold tolerance acquisition in broilers.

An experiment was conducted to evaluate the effects of a periodically low eggshell temperature exposure during incubation and dietary supplementation of arginine on performance, ascites incidence, and cold tolerance acquisition in broilers. A total of 2,400 hatching eggs were randomly assigned to 2 treatment groups (16 replicates of 75 eggs per treatment). The eggs were incubated at a constant eggshell temperature (EST) of 37.8ºC throughout the incubation period (CON) or were periodically exposed to 15°C for one hour on days 11, 13, 15, and 17 of incubation and the EST was measured (periodical low EST; PLE). After hatching, 240 one-day-old male broiler chicks from both treatment groups were reared for 42 d with or without dietary arginine supplementation in a completely randomized design with a 2 × 2 factorial arrangement. In order to induce ascites, all chicks were exposed to a 15°C room temperature from 14 d onwards. Results showed that second grade chicks and yolk sac weight were decreased, and final body weight was increased in the PLE group. Ascites mortality rate was decreased only in the PLE group and dietary arginine supplementation had no apparent effect. In the PLE group, the packed cell volume (PCV) percentage and red blood cell (RBC) count were decreased. In conclusion, the results showed that the PLE treatment during incubation was associated with improved hatchability, chick quality, and productive performance of broilers and decreased ascites incidence during post hatch cold exposure. Dietary arginine supplementation had no beneficial effects in cold exposed broilers.

Dietary supplementation with sodium bicarbonate improves calcium absorption and eggshell quality of laying hens during peak production.

The advantage of supplemental sodium bicarbonate (NaHCO3) on eggshell quality in laying hens changes with age. Besides increasing calcium (Ca) secretion in the eggshell gland, it may improve Ca absorption in the intestine or kidney. Hy-Line Brown layers (n = 384), 25 weeks of age, were allocated to two treatment groups in two experiments, each of which included 4 replicates of 24 hens. Hens were fed a basal diet (control) or the basal diet containing 3 g NaHCO3 g/kg for 50 or 20 weeks in Experiment 1 or 2, respectively. A 24-h continuous lighting regimen was used to allow hens to consume the dietary supplements during the period of active eggshell formation. In Experiment 1, particularly from 25 to 50 weeks of age, and in Experiment 2, NaHCO3 supplementation favoured hen-d egg production at the expense of lower egg weight. The increased eggshell thickness should have nothing to do with the additional eggshell formation, because of the unchanged egg mass and daily eggshell calcification. At 35 weeks of age in both experiments, NaHCO3 supplementation increased duodenal expression of calbindin-d28k (CaBP-D28k) protein, contributing to higher Ca retention and balance. From 50 to 75 weeks of age in Experiment 1, the hens had little response to NaHCO3 supplementation and showed a negative trend on eggshell thickness and strength. It is concluded that dietary supplementation with 3 g NaHCO3 g/kg improves Ca absorption and eggshell quality of laying hens during the peak but not late production period, with the introduction of continuous lighting.

Effects of age and dietary soybean oil level on eggshell quality, bone strength and blood biochemistry in laying hens.

The objective of the study was to investigate the differences in eggshell quality, bone quality and serum bone biochemistry markers associated with changes in age and dietary soybean oil levels in laying hens. A total of 54, 19-week-old Hy-Line Brown laying hens were housed in 18 battery cages (3 birds/cage) and randomly divided into three diet treatments for 90 d: control-fat (CF, 1.9% soybean oil), moderate-fat (MF, 7% soybean oil) and high-fat (HF, 10% soybean oil). The hens' body weights (BW), egg production, egg weights, eggshell thickness and femoral diameter were higher at d 90 than at d 60 or d 30. Meanwhile, feed intake, relative bone weights, all bone strength parameters and serum Ca were lower at d 90 or 60 than at d 30. Compared to the CF hens, the feed intake, BW, abdominal fat pad weights and serum alkaline phosphatase activity were elevated in MF or HF hens. The eggshell thickness, relative femoral and tibial weight, femoral stiffness, femoral modulus, tibial mixed force and serum calcium and phosphorus levels were lower in MF or HF hens than CF hens. These findings suggest that bone loss in caged hens starts from an early stage of the laying period, and dietary oil (particularly with diets over 10% soybean oil) has harmful effects on eggshell quality, bone strength and bone mineralisation from an early stage of the laying period.