Effects of low dietary calcium and lipopolysaccharide challenges on production performance, eggshell quality, and bone metabolism of laying hens.

Dietary calcium supply is essential for bone development and egg production in laying hens. This study investigated the effects of low dietary calcium and lipopolysaccharide (LPS) induced immune challenge in aged laying hens. A total of thirty-two Hy-Line Brown laying hens at 80 weeks old with an average laying rate of 62% were randomly divided into two groups and fed a normal calcium diet (3.57% Ca, NCA) or low calcium diet (2.08% Ca, LCA). At 88 weeks, the experiment was designed using a 2 × 2 factorial arrangement, and hens were intraperitoneally injected with saline (SAL) or LPS (0.5 mg/kg, 0.5 mg/kg, or 1.5 mg/kg body weight) once every 48 h intervals over 5 days. Production performance, egg quality, and bone physiology were evaluated. Results showed that LPS challenge decreased the hen-day egg production, egg mass, and eggshell traits (p < 0.05), but increased (p < 0.05) the calcium content of the tibia compared to SAL-injected hens. LCA diet decreased (p < 0.05) the hen-day egg production, and eggshell traits such as weight, percentage, strength, and thickness compared to the NCA diet. LCA diet increased the serum alkaline phosphatase (ALP) activity (p < 0.01) and tibial expression of ALP (p < 0.05) compared to NCA diet. LPS injection suppressed both the serum ALP activity (p < 0.05) and tibial expression of ALP (p < 0.001) compared to SAL injection. Furthermore, LPS injection increased (p < 0.05) the expression of both pro and anti-inflammatory cytokines in the spleen and tibia. The expression of cathepsin K ( Cts K ) and matrix metalloproteinase 9 ( MMP-9 ) were downregulated by LPS injection (p < 0.001). Broken and shell-less egg production and calcium content of eggshell, as well as tibial mRNA expression of osteocalcin ( Ocn ), tumor necrosis factor-alpha ( TNF-α ) and tartrate-resistant acid phosphatase ( TRAP ) were affected by the interaction (p < 0.05) of diet and injection. Therefore, this study demonstrated that to certain extents, low dietary calcium and LPS challenge dysregulated bone homeostasis and metabolism, with detrimental effects on the performance and eggshell quality of aged laying hens.

Dietary Super-Doses of Cholecalciferol Fed to Aged Laying Hens Illustrates Limitation of 24,25-Dihydroxycholecalciferol Conversion.

Background: Older humans taking high concentrations of vitamin D3 supplementation for a prolonged time may be at risk of vitamin D toxicity. It is unclear how dietary super-doses (10,000 times greater than the requirement) can affect vitamin D3 status in aged animals. Aged laying hens could be a model to compare vitamin D3 supplementation effects with women in peri- or postmenopausal stages of life. Objectives: We investigated the dietary super-dose impacts of cholecalciferol (vitamin D3) on vitamin D3 status in aged laying hens in production. Methods: Forty-eight 68-wk-old Hy-Line Brown laying hens were individually housed in cages with 8 hens per dietary treatment for 11 wk. Hens were randomly assigned to 1 of 6 treatment groups of dietary vitamin D3 supplementation and consumed ad libitum. Supplementation concentrations were 400, 800, 7400, 14,000, 20,000, and 36,000 IU D3/kg of feed. At the end of the study, all hens were sacrificed, and tissue samples and feces were collected. Plasma and egg yolk vitamin D3 metabolites, calcium and phosphorus composition of eggshells, ileal digesta, and feces were measured. Duodenal, ileal, liver, and kidney gene expression levels were also measured. Results: We observed that increasing dietary vitamin D3 increased plasma vitamin D3 and egg yolk vitamin D3 (P < 0.0001 for both sites). We also observed an increase in plasma 24,25-dihydroxycholecalciferol as dietary vitamin D3 concentrations increased (P < 0.0001). The plasma 25-hydroxycholecalciferol:24,25-dihydroxycholecalciferol ratio exhibited an asymptotic relationship starting at the 14,000 IU/kg D3 treatment. Conclusions: Dietary super-doses of vitamin D3 led to greater plasma and egg yolk vitamin D3 concentrations, which shows that aged laying hens can deposit excess vitamin D3 in egg yolk. We suggest future research should explore how 24-hydroxylation mechanisms are affected by vitamin D3 supplementation. Further understanding of 24-hydroxylation can help ascertain ways to reduce the risk of vitamin D toxicity.

Long-term supplementation of genistein improves immune homeostasis in the aged gut and extends the laying cycle of aged laying hens.

Aging is associated with alterations in gut function, including intestinal inflammation, leaky gut, and impaired epithelial regeneration. Rejuvenating the aged gut is imperative to extend the laying cycle of aged laying hens. Genistein is known to have beneficial effects on age-related diseases, but its precise role in homeostasis of the aged gut of laying hens remains to be elucidated. In this study, 160 45-wk-old Hyline Brown laying hens were continuously fed a basal diet or a diet supplemented with 40 mg/kg genistein until they reached 100 wk of age. The results revealed that long-term genistein supplementation led to an improvement in the egg production rate and feed conversion ratio, as well as an increase in egg quality. Moreover, the expression levels of senescence markers, such as ß-galactosidase, P16, and P21, were decreased in the gut of genistein-treated aged laying hens. Furthermore, genistein ameliorated gut dysfunctions, such as intestinal inflammation, leaky gut, and impaired epithelial regeneration. Treg cell-derived IL-10 plays a crucial role in the genistein-induced regulation of age-related intestinal inflammation. This study demonstrates that long-term consumption of genistein improves homeostasis in the aged gut and extends the laying cycle of aged laying hens. Moreover, the link between genistein and Treg cells provides a rationale for dietary intervention against age-associated gut dysfunction.

Effects of puerarin as a feed additive on the laying performance, egg quality, endocrine hormones, antioxidant capacity, and intestinal morphology of aged laying hens.

The aim of this study was to investigate the effects of puerarin (Pue), a phytoestrogen, on the production performance, egg quality, endocrine hormones, antioxidant capacity, and intestinal morphology in aged laying hens. A total of 180 Hy-Line Brown hens aged 480 d were randomly divided into 4 groups (n = 45 per group) and fed 0, 200, 400, and 800 mg/kg of Pue (Con, L-Pue, M-Pue, and H-Pue, respectively) during a 42-d experiment. Compared with the Con treatment, supplementation with H-Pue improved laying performance and egg quality by significantly increasing egg production, average egg weight, albumen height, yolk weight, and Haugh unit (P < 0.05) while decreasing the feed conversion ratio (P < 0.05). A diet supplemented with H-Pue significantly decreasing serum total triglycerides, total cholesterol, and low-density lipoprotein cholesterol, alanine aminotransferase (P < 0.05), and significantly increasing serum levels of follicle-stimulating hormone, luteinizing hormone and progesterone (P < 0.05). Antioxidant activity was improved by significantly increasing the activity of total antioxidant capacity, glutathione peroxidase and catalase but decreasing malondialdehyde levels in serum, jejunum, and ileum (P < 0.05), and superoxide dismutase activity exhibited a significantly increase in the jejunum and ileum (P < 0.05). Villus height and the ratio of villus height to crypt depth (P < 0.05) were significantly increased in the jejunum and ileum. In the jejunal and ileal mucosa, the three treatment groups increased the mRNA expression levels of Claudin-1 and Claudin-2 compared with Con (P < 0.05), and no significant effect was observed on the expression of Occludin and ZO-1. The results showed that dietary supplementation with Pue could improve the laying performance, egg quality, antioxidant capacity, hormonal profile, and intestinal morphology of aged laying hens.

Effects of supplemental vitamin A on reproduction and antioxidative status of aged laying hens, and growth, blood indices and immunity of their offspring.

The purpose of this investigation was to evaluate the impacts of vitamin A (VA) supplementation in feed at levels of 0 (control), 2,000, 4,000, 6,000, and 8,000 IU VA/kg diet on the reproductive efficiency and antioxidative properties of aged Sinai laying hens at 52 wk of age (n = 300 females and 30 males) in 6 replicates (10 females + 1 male/replicate). As well as blood biochemical indicators, carcass characteristics, growth performance, immunity, and the antioxidative status of their chicks. Results showed that diets supplemented with 2,000 or 6,000 IU/kg of VA increased fertility rate and decreased early embryonic mortality (P < 0.05). Increasing VA from 4,000 to 6,000 IU/kg significantly boosted hatchability rates. All VA levels significantly enhanced glutathione peroxidase (GPx) and reduced malondialdehyde (MDA) and late embryonic mortality. In the shell gland, dietary supplementation of 6,000 or 8,000 IU/kg of VA enhanced actions of GPx actions, catalase (CAT), and superoxide dismutase (SOD). In hatched chicks, all VA levels boosted (P < 0.05) hemoglobin, red blood cell count, and serum concentration of total proteins and IgA while decreasing eosinophils percentage and aspartate aminotransferase activity (AST) concentration. Dietary VA supplementations from 4,000 to 8,000 IU/kg improved lymphocytes, serum total antioxidant capacity (TAC), SOD, and IgM, while decreasing heterophils, heterophils/lymphocytes ratio, and creatinine in hatched chicks. Serum triglyceride concentration was reduced by adding 6,000 or 8,000 IU/kg of VA, while globulin and high-density lipoprotein concentrations were heightened only by 8,000 IU/kg of VA. It could be concluded that the dietary supplementation of VA (6,000 IU/kg) improved reproductive efficiency and antioxidative status in the liver and the shell gland of aged laying hens and improved hemato-biochemicals parameters, antioxidative status, and immunity of their offspring.

Effect of replacing dicalcium phosphate with mono-dicalcium phosphate to supplement phosphorus on laying performance, phosphorus-calcium metabolism and bone metabolism of aged laying hens.

The objective of this study was to evaluate the effect of replacing dicalcium phosphate (DCP) with mono-dicalcium phosphate (MDCP) to formulate low-phosphorus (P) diets on laying performance, egg quality, phosphorus-calcium metabolism, and bone metabolism of 69-78-week-old aged laying hens. Hy-Line Brown laying hens (n = 1,350, 69 weeks old) were randomly assigned to six treatments, each with five replicates of 45 hens. A corn-soybean meal-based diet was formulated to contain 0.12% non-phytate phosphorus (NPP), 3.81% calcium (Ca), and 1,470 FTU/kg phytase. The control group (CON) was supplemented with DCP inorganic phosphorus (Pi) at the NPP level of 0.20% (dietary NPP levels of 0.32%). Test groups (T1-T5) were supplemented with MDCP Pi at NPP levels of 0.07%, 0.11%, 0.15%, 0.18, and 0.20% (dietary NPP levels of 0.19, 0.23, 0.27, 0.30, and 0.32%, respectively). Calcium carbonate levels were adjusted to ensure all experimental diets contained the same Ca levels (3.81%). The feeding trial lasted 10 weeks, with hens increasing in age from 69 to 78 weeks. When supplemented with 1,470 FTU/kg phytase, extra DCP Pi or MDCP Pi did not affect (p > 0.05) laying performance (day laying rate, average egg weight, feed intake, feed-to-egg mass ratio, broken egg rate), egg quality (eggshell strength, albumen height, haugh units), or serum P, Ca, copper (Cu), iron (Fe), zinc (Zn), and manganese (Mn) levels. However, when laying hens were fed MDCP Pi (NPP levels of 0.07 to 0.20%), yolk color improved (p = 0.0148). The tibia breaking strength was significantly higher (p < 0.05) in the 0.18 and 0.20% NPP MDCP Pi groups than in the 0.20% NPP DCP Pi group. The breaking strength, Ca content, and P content of tibia in 0.11% and 0.15% NPP MDCP Pi hens were not significantly (p > 0.05) different from those in 0.20% NPP DCP Pi hens. Hens fed 0.07% NPP MDCP Pi had higher (p < 0.01) serum levels of osteoprotegerin (OPG), type-I collagen c-telopeptide (CTX-I), and tartrate-resistant acid phosphatase 5b (TRACP-5b) than those in all other groups. Serum levels of TRACP-5b and CTX-I in the 0.11% and 0.15% NPP MDCP Pi group were significantly lower than those in 0.18 and 0.20% NPP MDCP Pi groups and the 0.20% NPP DCP Pi group (p < 0.0001). Hens fed 0.07% and 0.11% NPP MDCP Pi had higher (p < 0.05) serum levels of parathyroid hormone (PTH) than those in all other groups. No differences were detected in serum calcitonin (CT), 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3), bone alkaline phosphatase (BAP), osteocalcin(OCN), and osteopontin (OPN) among all groups (p > 0.05). The expression of P transporters type IIa Na/Pi cotransporter (NaPi-IIa) in 0.11% and 0.15% NPP MDCP Pi hens were higher than those in 0.20% NPP MDCP Pi group and 0.20% NPP DCP Pi group (p < 0.05). The results indicated that both renal P reabsorption and bone resorption were involved in adapting to a low-P diet. In summary, when MDCP was used instead of DCP to supplement P, NPP levels could be reduced to 0.11% (dietary NPP level of 0.23%) without negative effects on laying performance and skeletal health of aged hens. In addition, MDCP was more beneficial than DCP for tibia quality. The results of the current study would provide references for the application of MDCP in low-P diets of aged laying hens.

Effects of Dietary Phytosterol Supplementation on the Productive Performance, Egg Quality, Length of Small Intestine, and Tibia Quality in Aged Laying Hens.

This study aimed at investigating the effects of phytosterols on the productive performance, egg quality, length of small intestine, and tibia quality in aged laying hens. A total of 960 Dawu Jinfeng commercial laying hens (75 weeks of age) were randomly assigned to three groups. Each group had 16 replicates and every replicate contained four cages (five birds/cage). The control group hens received the basal diet without phytosterols. The hens in the experimental groups received a diet containing phytosterols at concentrations of 20 mg/kg and 40 mg/kg for 7 weeks. The results showed that phytosterols had a linearly increasing effect on egg weight, eggshell surface area, albumen height, and haugh unit at week 5 of experiment (p < 0.05). Supplemental phytosterols linearly and quadratically increased eggshell thickness (p < 0.05). At week 7 of the experiment, dietary supplementation of phytosterols linearly increased egg weight and eggshell weight (p < 0.05). Supplementation of 20 mg/kg, but not 40 mg/kg, phytosterols increased the length of the small intestine. However, dietary phytosterols had no effect on the laying rate, mortality, or liver index (p > 0.1). The results of tibia quality detected by micro-CT also showed no difference in the treatment of phytosterols. Therefore, supplementation with 20 mg/kg phytosterols in the diet improves egg quality and increases the length of small intestine, but has no effects on the quality of the tibia.

Effect of dietary concentrations of metabolizable energy and neutral detergent fiber on productive performance, egg quality, fatty liver incidence, and hepatic fatty acid metabolism in aged laying hens.

The objective of the current experiment was to investigate the effect of dietary concentrations of ME and neutral detergent fiber (NDF) on productive performance, egg quality, fatty liver incidence, and hepatic fatty acid metabolism in aged laying hens. A total of three hundred twenty 75-wk-old Hy-Line Brown laying hens were allotted to 1 of 4 dietary treatments with 8 replicates. Each replicate consisted of 10 consecutive cages with 1 hen per cage. The experiment was conducted using a completely randomized design with 2 × 2 factorial arrangement consisting of 2 levels of ME (normal [commercially recommended AMEn levels; 2,730 kcal/kg] and low [50 kcal/kg reduction in AMEn; 2,680 kcal/kg]) and 2 levels of NDF (low [9.01 and 9.61%; normal-ME and low-ME diets, respectively] and high [12.57 and 13.42%; normal-ME and low-ME diets, respectively]) in the diet. The diets and water were provided to hens on an ad libitum basis for 12 wk. Results indicated that no interactions between dietary concentrations of ME and NDF were observed for all measurements except for egg yolk color, eggshell thickness, and 2 hepatic gene expressions (i.e., carnitine palmitoyl transferase 1A and malic enzyme). For the main effects, increasing NDF concentrations in diets increased (P < 0.05) feed intake without affecting other productive performance. Hens fed normal-ME and high-NDF diets showed the darkest (P < 0.05) egg yolk color among those fed treatment diets, showing an interaction (P < 0.05). Increasing NDF concentrations in low-ME diets did not influence eggshell thickness, but those in normal-ME diets increased eggshell thickness in laying hens, showing an interaction (P < 0.05). For the main effects, increasing concentrations of dietary NDF or ME reduced (P < 0.05) hepatic fat concentrations with decreasing expressions in several genes related to fatty acid synthesis. In conclusion, increasing NDF concentrations in commercially-recommended ME diets decrease hepatic fat concentrations in aged laying hens, and therefore, may have a preventative effect on the fatty liver development in aged laying hens.

Dietary supplementation with astaxanthin alleviates ovarian aging in aged laying hens by enhancing antioxidant capacity and increasing reproductive hormones.

We investigated the effects of astaxanthin supplementation on the egg quality, antioxidant capacity, and ovarian aging of aged laying hens. Six groups of 68-wk-old Hy-line brown laying hens with six replications each, fifteen chickens in each replicate were fed for 12 wk. The control group was fed a basal diet, the positive control group was fed the basal diet supplemented with 100 mg/kg vitamin E, and the experimental groups were fed the basal diet supplemented with 15 mg/kg, 30 mg/kg, 45 mg/kg, or 60 mg/kg astaxanthin (Ax15, Ax30, Ax45, and Ax60, respectively). The results showed that astaxanthin accumulated in the egg yolks and improved egg yolk color (P < 0.01) and Haugh unit (P < 0.05). Compared with the control group, the experimental groups a higher number of follicles in the ovary and a lower rate of atresia (P < 0.01). Astaxanthin increased the expression of nuclear factor e2-related factor 2 (NRF2) in the ovary (P < 0.05), enhanced the antioxidant capacity of aged laying hens (P < 0.05), and reduced cellular apoptosis (P < 0.05). In addition, astaxanthin improved serum reproductive hormone levels (follicle-stimulating hormone, luteinizing hormone, and progesterone) (P < 0.05) with a maximum value observed in Ax60. However, astaxanthin had no effects on estrogen level (P > 0.05). The expression of FSHR and CYP11A1 increased in the follicular granulosa cells (P < 0.05). Therefore, astaxanthin prevented ovarian aging by improving the antioxidant capacity of laying hens and promoting the production of reproductive hormones. The declining reproductive performance of laying hens in the late laying period may be improved with astaxanthin supplementation.

Transcriptomic analysis of the liver in aged laying hens with different eggshell strength.

Eggshell is composed of a very ordered and mineralized structure and is important for egg quality. Eggshell strength is particularly important because of its direct association with economic outcomes and egg safety. Various factors related to laying hens and their environment affects eggshell strength. However, the molecular mechanisms of liver functions related to decreased eggshell strength of aged laying hens are largely unknown. Therefore, this study aimed to identify potential factors affecting eggshell strength in aged laying hens at the hepatic transcriptomic level. A total of five hundred 92-wk-old Hy-line Brown laying hens were screened to select those exhibiting the greatest variation in eggshell strength. Based on the final eggshell strength, 12 hens producing eggs with strong eggshell strength (SES) and weak eggshell strength (WES) were finally selected (n = 6) for liver tissue sampling. The RNA-sequencing was performed to identify differentially expressed genes (DEGs) between the 2 groups. We identified a total of 2,084 DEGs, of which 1,358 genes were upregulated and 726 genes were downregulated in the WES group compared with SES group. According to the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, the DEGs indicated the mammalian target of rapamycin signaling pathway, the Janus kinase-signal transducer and activator of transcription pathway, the mitogen­activated protein kinase signaling pathway, and the insulin resistance pathways. Genes related to fatty liver disease were upregulated in WES group compared with SES group. In addition, expression of several genes associated with oxidative stress and bone resorption activity was altered in aged laying hens with different eggshell strength. Overall, these findings contribute to the identification of genes involved in different intensity of eggshell strength, enabling more understanding of the hepatic molecular mechanism underlying in decreased eggshell strength of aged laying hens.

Microbiome and ileum transcriptome revealed the boosting effects of selenium yeast on egg production in aged laying hens.

The declines in laying performance during the late production period have adverse effects on the length of the production cycle. Improving the nutrition of laying hens is a crucial measure to reverse this declination. This study investigated the effect of selenium yeast (SY) on egg production, ileal gene expression and microbiota, as well as elucidating their associations in aged laying hens. A total of 375 Jinghong laying hens at 76 weeks old were randomly assigned into 5 dietary treatments, which included a selenium-deficient basal diet based on corn-soybean meal, and dietary supplementation of SY at 0.15, 0.30 and 0.45 mg/kg, and sodium selenite at 0.45 mg/kg. The results showed that SY ameliorated the depression in aged laying performance in the 0.30 mg/kg group (P < 0.01). Selenium yeast significantly increased ileum selenium concentration (P < 0.05), and SY groups had higher selenium deposition efficiency than the sodium selenite group. Functional enrichment and Short Time-series Expression Miner (STEM) analysis indicated that SY activated metabolic progress (e.g., glycerolipid metabolism, glycerophospholipid metabolism, and fatty acid metabolism), immune response and oxidative stress response. Four hub genes including thioredoxin reductase 1 (TXNRD1), dihydrolipoamide dehydrogenase (DLD), integrin linked kinase (ILK) and leucine zipper tumor suppressor 2 (LZTS2) were involved in intestinal metabolism which was closely associated with selenium deposition/status. Moreover, the relative abundance of Veillonella, Turicibacter and Lactobacillus was significantly increased, but the relative abundance of Stenotrophomonas was significantly decreased by SY supplementation. Multi-omics data integration and Canonical correspondence analysis (CCA) showed that both the ileum selenium content and the laying rate were highly correlated with pathways and bacteria enriched in metabolism and immune response. Meanwhile, the "switched on" gene prostate stem cell antigen (PSCA) had a positive relationship with Veillonella and a negative relationship with the opportunistic pathogens Stenotrophomonas. Overall, our study offered insight for the further exploration of the role of SY on boosting egg production and balancing ileum intestinal flora in aged laying hens.

Mitochondrial transcription factor A induces the declined mitochondrial biogenesis correlative with depigmentation of brown eggshell in aged laying hens.

Eggshell color is an important characteristic for poultry eggs. Eggs from aged hens usually have poor shell color that is unacceptable for the table egg market. The objective of this study was to examine effects of pigment synthesis and mitochondrial biogenesis on brown eggshell color of aged laying hens. In this trial, 8 hens laying eggs with darker shell color and 8 hens laying eggs with lighter shell color were selected from 300 62-week-old Hy-Line brown-egg laying hens. Results showed that egg weight (P < 0.05), eggshell weight (P < 0.01), protoporphyrin IX (Pp IX) content of the eggshell and the shell gland (P < 0.001), and biliverdin content of the shell gland (P < 0.001) were significantly declined in the light-shell group compared with the dark-shell group. Relative mRNA expression of δ-aminolevulinic acid synthase1 (ALAS1) (P < 0.05), coproporphyrinogen oxidase (P < 0.01), ATP-binding cassette transporter ABCG2 (P < 0.01), and mitochondrial transcription factor A (P < 0.05) was reduced in hens laying lighter brown eggshell. Moreover relative mRNA expression of mitochondrial DNA copy number (P < 0.01), mitochondrial NADH dehydrogenase subunit 4 (P < 0.05), mitochondrial ATP synthase F0 subunit 8 (P < 0.05), and mitochondrial cytochrome c oxidase 1 (P < 0.01) was significantly decreased in the shell gland of the light-shell group. In addition, NAD+ contents of the shell gland were increased in the dark-shell group (P < 0.01). Brown eggshell depigmentation is a result of decreased Pp IX content in the eggshell and the shell gland. Decreased mitochondrial biogenesis may contribute to the depigmentation of brown eggshell by targeting ALAS1 and ALAS1-mediated Pp IX biosynthesis.

Transcriptomic analysis of the liver in aged laying hens with different intensity of brown eggshell color.

OBJECTIVE: Eggshell color is an important indicator of egg quality for consumers, especially for brown eggs. Various factors related to laying hens and their environment affect brown eggshell coloration. However, there have been no studies investigating hepatic functions of laying hens with variable intensity of brown eggshell color. Therefore, this study was aimed to identify potential factors affecting brown eggshell coloration in aged laying hens at the hepatic transcriptomic level. METHODS: Five hundred 92-wk-old Hy-line Brown laying hens were screened to select laying hens with different intensity of brown eggshell color based on eggshell color fans. Based on eggshell color scores, hens with dark brown eggshells (DBE; eggshell color fan score = 14.8) and hens with light brown eggshells (LBE; eggshell color fan score = 9.7) were finally selected for the liver sampling. We performed RNA-seq analysis using the liver samples through the paired-end sequencing libraries. Differentially expressed genes (DEGs) profiling was carried out to identify their biological meaning by bioinformatics. RESULTS: A total of 290 DEGs were identified with 196 being up-regulated and 94 being down-regulated in DBE groups as compared to LBE groups. The Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that these DEGs belong to several biological pathways including herpes simplex infection (toll-like receptor 3 [TLR3], cyclin-dependent kinase 1, etc.) and influenza A (TLR3, radical S-adenosyl methionine domain containing 2, myxovirus [influenza virus] resistance 1, etc.). Genes related to stress response (ceremide kinase like) and nutrient metabolism (phosphoenolpyruvate carboxy-kinase 1, methylmalonic aciduria [cobalamin deficiency] cblB type, glycine receptor alpha 2, solute carrier family 7 member 11, etc.) were also identified to be differentially expressed. CONCLUSION: The current results provide new insights regarding hepatic molecular functions related to different intensity of brown eggshell color in aged laying hens. These insights will contribute to future studies aiming to optimize brown eggshell coloration in aged laying hens.

Yeast culture promotes the production of aged laying hens by improving intestinal digestive enzyme activities and the intestinal health status.

Yeast culture (YC) positively affects the performance of laying hens. The purpose of the present study was to explore the underlying mechanism for the YC-mediated performance improvement. Sixty 67-week-old Hy-Line Brown laying hens were randomly allocated into 2 experimental groups with 5 replicates of 6 birds each. One group was fed a control diet, whereas the other received the control diet supplemented with YC at 3.0 g/kg; treatment lasted for 8 wk. The results showed that dietary YC supplementation increased (P < 0.05) the total egg weight (11.2-13.6%) and egg-laying rate (13.0-13.5%) but decreased (P < 0.05) the feed/egg ratio by 9.3 to 11.0% during weeks 5 to 6 and 7 to 8 compared with the control. However, egg quality, including eggshell strength, eggshell thickness, egg weight, albumen height, egg yolk color, and Haugh unit, was not affected (P > 0.05) by YC supplementation. Furthermore, dietary YC supplementation increased (P < 0.05) chymotrypsin and ɑ-amylase activities by 54.8 to 62.5% in the duodenal chyme and reduced (P < 0.05) plasma endotoxin by 44.1%. YC dietary supplementation also upregulated (P < 0.05) the mRNA levels of intestinal barrier-related genes (occludin and claudin 1) and antimicrobial peptides genes (ß-defensin 1 and 7 and cathelicidin 1 and 3) in the duodenum or jejunum compared with the control. In conclusion, dietary YC supplementation improved the performance of aged laying hens, potentially through the upregulation of intestinal digestive enzyme activities and intestinal health-related gene expression.

New insights into the role of dietary marine-derived polysaccharides on productive performance, egg quality, antioxidant capacity, and jejunal morphology in late-phase laying hens.

The present study was conducted to evaluate the effects of dietary marine-derived polysaccharides (MDP) from seaweed Enteromorpha on productive performance, egg quality, antioxidant capacity, and jejunal morphology in late-phase laying hens. A total of 240 Lohmann white laying hens (62 wk of age) were assigned to 4 dietary treatments that included MDP at concentrations of 0, 1,000, 2,500, and 5,000 mg/kg for 6 wk. Each treatment had 6 replicates with 5 cages (2 birds/cage). The results showed that dietary MDP quadratically improved egg production (P < 0.05) during 5 to 6 wk and 1 to 6 wk. There was a linear reduction in cracked egg rate (P < 0.05) with dietary MDP levels increased during 3 to 4 wk and 1 to 6 wk. After 4 wk of feeding trial, the egg shell thickness, yolk color, and Haugh unit showed a linear increase (P < 0.05) in response to increasing dietary MDP levels. Besides, the egg shell breaking strength, egg shell thickness, yolk color, and Haugh unit were improved linearly (P < 0.05) by dietary MDP at the end of the experiment. Moreover, dietary MDP showed a linear and quadratic reduction in serum malondialdehyde (MDA) content (P < 0.05) at the end of third week. At the end of experiment, the activity of total superoxide dismutase in serum was increased quadratically (P < 0.05) by dietary MDP, and dietary MDP quadratically improved the liver catalase (CAT) activity (P < 0.05) and linearly enhanced jejunal CAT activity (P < 0.05), whereas linearly decreased jejunal MDA concentration (P < 0.05). Furthermore, supplemental MDP linearly improved the villus height (P < 0.05) and quadratically increased villus height/crypt depth ratio (P < 0.05) of jejunum. However, dietary MDP had no effect on jejunal trypsin, amylase, and protease activity (P > 0.10). Taken together, these findings provided new insights into the role of MDP in improving the productive performance, egg quality, antioxidant capacity, and jejunal morphology of late-phase laying hens.

Effects of the Methionine Hydroxyl Analogue Chelate Zinc on Antioxidant Capacity and Liver Metabolism Using 1H-NMR-Based Metabolomics in Aged Laying Hens.

This study aimed to investigate the effects of different levels of methionine hydroxyl analogue chelated zinc (MHA-Zn) on antioxidant capacity and liver metabolism of aged laying hens. A total of 960 57-week-old layers were fed a basal diet (Zn: 35.08 mg/kg) without extra zinc for two weeks, and then allocated to four treatments consisting of eight replicates of 30 birds each for 14 weeks. Four levels of Zn (zinc sulfate (ZnSO4): 80 mg/kg; MHA-Zn: 20, 40, 80 mg/kg) were added to the diet. The results indicated that compared with inorganic zinc, organic zinc of 80 mg/kg has a significant advantage in improving the antioxidant capacity of aged hens, which increased the level of Cu/Zn-superoxide dismutase (SOD) and the total antioxidant capacity (T-AOC) in the serum and liver, and reduced the concentration of malondialdehyde (MDA) of laying hens. The serum albumen composition was significantly modified, meanwhile, the level of total protein, globulin, and urea increased remarkably, whereas serum glutamic-oxaloacetic transaminase decreased notably in 80 mg/kg MHA-Zn groups. Compared with the 20 mg/kg MHA-Zn group, the metabolic profile of 40 and 80 mg/kg MHA-Zn groups was higher than that of the inorganic zinc group. Furthermore, integrated key metabolic pathway analysis showed that 40 and 80 mg/kg MHA-Zn groups participated in the regulation of glutathione metabolism, glycine, serine, and threonine metabolism. Therefore, this study suggests that 40 and 80 mg/kg supplementation of MHA-Zn can increase the activity of Cu/Zn-SOD and T-AOC and decrease MDA; additionally the 80 mg/kg MHA-Zn group has better antioxidant capacity. Meanwhile, the enhanced MHA-Zn promoted methionine (Met) synthesis and protein metabolism.

Analysis of excreta bacterial community after forced molting in aged laying hens.

OBJECTIVE: As laying hens become aged, laying performance and egg quality are generally impaired. One of the practical methods to rejuvenate production and egg quality of aged laying hens with decreasing productivity is a forced molting. However, the changes in intestinal microbiota after forced molting of aged hens are not clearly known. The aim of the present study was to analyze the changes in excreta bacterial communities after forced molting of aged laying hens. METHODS: A total of one hundred 66-wk-old Hy-Line Brown laying hens were induced to molt by a 2-d water removal and an 11-d fasting until egg production completely ceased. The excreta samples of 16 hens with similar body weight were collected before and immediately after molting. Excreta bacterial communities were analyzed by high-throughput sequencing of bacterial 16S rRNA genes. RESULTS: Bacteroidetes, Firmicutes, and Proteobacteria were the three major bacterial phyla in pre-molting and immediate post-molting hens, accounting for more than 98.0%. Lactobacillus genus had relatively high abundance in both group, but decreased by molting (62.3% in pre-molting and 24.9% in post-molting hens). Moreover, pathogenic bacteria such as Enterococcus cecorum and Escherichia coli were more abundant in immediate post-molting hens than in pre-molting hens. Forced molting influenced the alpha diversity, with higher Chao1 (p = 0.012), phylogenetic diversity whole tree (p = 0.014), observed operational taxonomic unit indices (p = 0.006), and Simpson indices (p<0.001), which indicated that forced molting increased excreta bacterial richness of aged laying hens. CONCLUSION: This study improves the current knowledge of bacterial community alterations in the excreta by forced molting in aged laying hens, which can provide increasing opportunity to develop novel dietary and management skills for improving the gastrointestinal health of aged laying hens after molting.

Effect of dietary ß-mannanase on productive performance, egg quality, and utilization of dietary energy and nutrients in aged laying hens raised under hot climatic conditions.

OBJECTIVE: The objective of this experiment was to investigate the effect of dietary ß-mannanase on productive performance, egg quality, and utilization of dietary energy and nutrients in aged laying hens raised under hot climatic conditions. METHODS: A total of 320 84-wk-old Hy-line Brown aged laying hens were allotted to one of four treatments with eight replicates in a completely randomized design. Two dietary treatments with high energy (HE; 2,800 kcal/kg nitrogen-corrected apparent metabolizable energy [AMEn]) and low energy (LE; 2,700 kcal/kg AMEn) were formulated. Two additional diets were prepared by adding 0.04% (MN4) or 0.08% ß-mannanase (MN8) to LE treatment diets. The feeding trial was conducted for 28 d, covering a period from July to August in South Korea. The average daily room temperature and relative humidity were 29.2°C and 83%, respectively. RESULTS: Productive performance, egg quality, and cloacal temperature were not influenced by dietary treatments. The measured AMEn values for MN8 diets were similar to those for HE diets, which were greater (p<0.05) than those for LE and MN4 diets. However, the AMEn values for MN8 diets did not differ from those for LE and MN4 diets. CONCLUSION: The addition of ß-mannanase to low energy diets increases energy values for diets fed to aged laying hens. However, this increase has little positive impacts on performance and egg quality. These results indicate that dietary ß-mannanase does not mitigate the heat stress of aged laying hens raised under hot climatic conditions.