Intestinal microbial metabolism has an important impact on the health of laying hens, and microbes are also important hosts for ARGs. However, the relationship between intestinal microbes and antibiotic resistance in laying hens is unclear. In this study, a slaughtering experiment, an in vitro fermentation experiment and a single-bacteria culture experiment were carried out, and metagenomic and metabolomic analyses were used to investigate the relationships between microbial metabolism and the antibiotic resistome in the cecum of laying hens. The results showed that there were different types of ARGs in the intestines of laying hens, and the risk scores of the ARGs tended to decrease with growth stage. A total of 1142 metagenome-assembled genomes (MAGs) were obtained, and Escherichia coli was found to be the dominant ARG host, carrying 62 ARGs. Metabolomics revealed that indole and its derivatives, such as indole-3-lactic acid, were negatively correlated with a variety of ARGs. Moreover, in vitro fermentation experiment and single-bacteria culture experiment demonstrated that indole-3-lactic acid reduced the abundance and risk of multiple ARGs in the intestine and inhibited the growth of the ARG host Escherichia coli. In the context of high concern about intestinal microbial metabolism and antibiotic resistance, this is the first study to focus on the relationship between intestinal microbial metabolism and antibiotic resistance in laying hens. These findings have important implications for healthy farming and antibiotic resistance control.

Climate change is increasingly manifesting in temperate regions. Laying hens are highly sensitive to heat stress and mitigation strategies should be implemented to reduce the negative effects. The goal of this experiment was to evaluate the effects of betaine in drinking water (0.55 g/L) and 4 h feed restriction during peak heat stress on laying performance, egg quality, blood gas parameters, body temperature (Tb), and oxidative stress in 2 different breeds of laying hens. Therefore, 448 ISA Brown hens (25 wk) and 448 Lohmann LSL classic laying hens (26 wk) were housed in 128 cages (7 hens/cage). Thermoneutral (TN) data was collected for 21 d before cyclic heat stress (HS) (21d; 32 ± 2°C; 6 h daily). During HS, hens were divided into 4 treatments: 1) feed restriction (FR), 2) betaine supplementation in drinking water (BET), 3) feed restriction and betaine supplementation in drinking water (FR-BET), or 4) control (CON). The effects were evaluated after 1 wk of HS (acute heat stress; AHS) and 3 wk of HS (chronic heat stress; CHS). Laying rate and egg mass (EM) diminished during CHS but decreased more in white than brown hens (2.78% and 1.94%; -1.57% and -0.81%, respectively; P = 0.004) and remained unaltered by BET or FR. During AHS, average daily feed intake (ADFI) increased compared to TN, but the increase was higher in white than brown hens (6.36% and 2.62%, respectively; P = 0.001). Egg shell quality deteriorated during AHS and CHS, but was most affected in white hens, FR or BET did not impact this. Blood pCO2, HCO3- and base excess significantly decreased during AHS and CHS, but pH and iCa were unaltered. Blood glucose increased in white hens during AHS compared to TN (P < 0.001), while plasma malondialdehyde increased in brown hens (P < 0.001). Results indicated that laying hens experienced HS, but breed differences were observed and white hens were generally most affected. FR affected feed conversion ratio negatively during CHS. However, FR and BET could not improve laying performance, egg quality, Tb, or blood parameters during HS.

Feed efficiency (FE) is an important economic factor in poultry production, and feed conversion ratio (FCR) is one of the most widely used measures of FE. Factors associated with FCR include genetics, the environment, and other factors. However, the mechanisms responsible for FCR in chickens are still less well appreciated. In this study, we examined the pattern changes of FCR, then delved into understanding the mechanisms behind these variations from both genetic and environmental perspectives. Most interestingly, the FCR at the front section of henhouse exhibited the lowest value. Further investigation revealed that laying rate in the high FCR (HFCR) group was lower than that in the low FCR (LFCR) group (P < 0.05). Cortisol, total antioxidant capacity (TAOC), and IgG levels in the LFCR group were significantly lower than those in the HFCR group (P < 0.05), while BUN level was significantly higher than that in the HFCR group (P < 0.05). We identified a total of 67 and 10 differentially expressed genes (DEGs) associated with FCR in ovarian and small intestine tissues, respectively. Functional enrichment analysis of DEGs revealed that they might affect FCR by modulating genes associated with salivary secretion, ferroptosis, and mineral absorption. Moreover, values for relative humidity (RH), air velocity (AV), PM2.5, ammonia (NH3), and carbon dioxide (CO2) in the LFCR group were significantly lower than those in the HFCR group (P < 0.05). Conversely, value for light intensity (LI) in the LFCR group was significantly higher than that in the HFCR group (P < 0.05). Correlation analysis revealed a positive correlation between FCR and RH, AV, PM2.5, NH3, and CO2, and a negative correlation with LI. Finally, the FCR prediction model was successfully constructed based on multiple environmental variables using the random forest algorithm, providing a valuable tool for predicting FCR in chickens.

Indigenous animal genetic resources should be preserved because of their well adaptation to the environment, their tolerance to low food availability and their sociocultural importance. The characterization of the quality of the products generated by heritage breeds may bring more arguments to encourage the raising of these animals. This study aimed at evaluating the egg performance and quality of Spanish indigenous Black Castellana (BC) breed as compared with a selected strain (Lohmann LSL-Classic). Four groups of 30 hens were arranged: 1) Lohmann hens fed a control diet; 2) BC hens fed the control diet; 3) Lohmann hens fed a diet including linseed at 70 g/kg (omega-3 diet); 4) BC hens fed the omega-3 diet. Egg production was higher by 12.3% for Lohmann hens but, since BC eggs were heavier by 15.4%, no effect of genetics was found on daily egg mass. Feed intake was higher by 5.0% for BC hens. Nonetheless, no difference was detected for feed conversion ratio. Eggshell was thicker by 6.78% in Lohmann eggs. Haugh units did not differ among freshly laid and stored eggs in Lohmann hens, whereas Haugh units decreased in stored BC eggs (80.5 vs. 76.7 vs. 72.3 at 0, 14, and 30 d of storage). Yolks of BC eggs contained less fat (57.5 vs. 60.8% DM), more protein (32.8 vs. 31.9% DM) and more cholecalciferol (1.25 vs. 1.22 μg/g DM), and showed lower proportion of saturated fatty acids (29.0 vs. 37.0%) and higher proportions of monounsaturated (45.7 vs. 39.6%) and polyunsaturated (25.2 vs. 23.4%) fatty acids. Feeding the omega-3 diet reduced the yolk proportions of saturated (32.5 vs. 33.5%) and monounsaturated (42.0 vs. 43.3%) fatty acids and increased those of polyunsaturated (25.4 vs. 23.2%) and ω-3 (7.05 vs. 2.42%) fatty acids. No effect due to genetics or diet was found on yolk color score or on yolk content in cholesterol, cobalamin, retinol and γ-tocopherol. This study represents the first exhaustive characterization of eggs from Spanish indigenous Black Castellana breed.

Micelle silymarin (MS) is recognized for its diverse range of beneficial properties, which encompass anti-inflammatory, antioxidant, hepatoprotective, and antidiabetic effects. The main objective of this study was to examine the effects of micelle silymarin on the performance, egg quality, blood profile, and absorption rate of silymarin in laying hens. In experiment 1: 288 Hy-Line brown laying hens, 28 wk old, were utilized for this experiment. The hens were randomly allocated into 3 dietary treatment groups, with each group comprising eight replicates of 12 hens, each housed in individual pens with access to feed and water. Over a 12-wk feeding trial, the hens were provided with a basal diet supplemented with different levels of MS: 0, 0.03, and 0.06%. In experiment 2: For this experiment, 192 Hy-Line Brown laying hens were divided into 2 dietary treatment groups, with each group comprising eight replications of 12 hens. The dietary treatments were: TRT1, basal diet + powder silymarin 4%; TRT2, basal diet + MS 4%. From the first experiment, the findings revealed that incorporating micelle silymarin (MS) into the hens\' diet significantly increased egg weight at wk 6 (P < 0.05). Similarly, at wk 12 and throughout the entire experiment, significant effects were observed on downgraded egg count, egg production, egg weight, and feed conversion ratio (FCR) (P < 0.05). Moreover, Haugh Units (HU) and albumen height showed a linear improvement (P < 0.05) at wk 4 with MS supplementation. Furthermore, there was a linear increase in egg yolk color, albumen height, and eggshell thickness at wk 8 with MS supplementation (P < 0.05). Furthermore, a layers-fed diet supplemented with MS showed a linear increase (P < 0.05) in HU, egg weight, yolk color, albumen height, eggshell strength, and eggshell thickness in wk 12. Regarding blood profile parameters, the study revealed linear reductions for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) (P < 0.05), whereas there was a tendency for albumin, triglyceride, and cholesterol (P < 0.10). In the second experiment, it was observed that the blood absorption rate of silymarin was higher in TRT2 compared to TRT1 at 2- and 4-h intervals following administration. In summary, increasing MS supplementation in the diet of laying hens enhanced egg production, egg quality, and blood profile. Additionally, silymarin absorption was higher in its micelle form than in its powder form.

To elucidate the regulatory mechanisms that impact variability in albumen quality of laying hens from the peak of lay to the late production phase. A 60-wk study was conducted on a cohort of 20,000 Hy-Line Brown laying hens from 20 to 80 wk old. Before commencement at 20 wk, the 10-wk-old hens were acclimatized for 10 wk. This study examined changes in albumen quality, serum, and liver antioxidant capacity, magnum morphology, and expression of albumen-protein-related genes in the magnum. To reduce sampling error, we collected eggs (n = 90) from pre-determined cages at every sampling point (5-wk intervals), and 8 hens were selected at 10-wk intervals for blood and tissue collection. Our findings revealed that age significantly affected most evaluated parameters. Albumen gel properties, including hardness, gumminess, and chewiness, increased significantly with age (P < 0.05). With the increasing of hens\' age from 20 to 80 wk, the albumen proportion of eggs was decreased, but eggshell proportion, yolk proportion, thick albumen proportion, thick to thin ratio, thick albumen solid content, albumen height, Haugh units (HU), and yolk color were increased and then decreased (P < 0.05). Compared to hens aged 20 to 60 wk, the hens (70-80 wk) had significantly reduced total antioxidant capacity (TAC) and glutathione levels (GSH) in the liver and lower serum TAC and superoxide dismutase levels (SOD) (P < 0.05). The magnum mucosal folds were highest in 40 to 60 wk-old hens, and the luminal diameter increased with age (P < 0.05). In the magnum, the mRNA expression levels for OVA, CPE, and NUP205 increased significantly between 30 and 40 wk, while FBN1 expression was higher between 30 and 50 wk (P < 0.05). At 70 to 80 wk, the expression of BRCA2 was significantly downregulated (P < 0.05). Albumen height, thick albumen proportion with protein secretion-related genes, enhanced antioxidant function, and luminal diameter correlated positively. However, the thick-to-thin albumen ratio negatively correlated with BRCA2, downregulated in aged laying hens. We used principal component and cluster analysis to deduce albumen quality changes during 3 phases: 25 to 35, 40 to 55, and 60 to 80 wk. The decline in albumen quality in aging hens is linked with decreased antioxidant capacity, magnum health, and downregulation of key genes involved in protein synthesis and secretion. These findings emphasize critical albumen quality changes in laying hens and suggest molecular pathways underlying age-related albumen quality alterations.

Vitamin D3 (25-hydroxyvitamin D3 (VD)) and vitamin E (VE) have proven to have immunomodulatory and antioxidant functions along with capacities to improve the reproductive function in chickens. Coccidiosis in laying hens at different stages of growth has been shown to negatively affect performance, immune response, and oxidative status, thus increasing the cost of production. A study was conducted to evaluate the influence of dietary VD or VE on performance, gut health, immune response, and oxidative status of laying hens at peak production. Laying hens (23 wk-of-age, n = 225) were randomly allocated into 5 treatment groups (n = 9 hens/replicate) with 5 replicate groups each: 1) unchallenged control (UC), 2) pair-fed control (PF), 3) challenged control (CC), 4) challenged control top-dressed with 5,000 IU of 25-hydroxyvitamin D3 (VD) per kg of diet, and 5) challenged control top-dressed with 100 IU of DL-α-tocopherol (VE). At 25 wk-of-age, hens grouped in CC, VD, and VE were challenged with mixed Eimeria spp. to induce coccidiosis. VD or VE supplemented hens did not impact bird body weight; however, egg production increased by 10.36% and 13.77%, respectively (P < 0.0001). Furthermore, the gut health of the hens was improved with either VD or VE supplementation, as indicated by lowered gut permeability and intestinal lesion scores (P < 0.05). VE significantly reduced the heterophil count (P = 0.0490) alongside numerically increasing the peripheral CD4+ and CD8+ T cells and monocyte counts (P > 0.05). Both VD or VE increased the TAC at 14 DPI compared to UC (P<0.05). Preliminary findings suggest that dietary VD or VE supplementation has the potential to improve gut health, modulate the immune response, and increase egg production in coccidiosis-infected laying hens.

Two trials were conducted to draw the phase-response curve of productive and immunological variables in heat-exposed layer chickens at different ages (71 to 130 d, and 211 to 270 d). Birds were acclimated to the following conditions for 60 d: constant optimal ambient temperature at 24°C and high ambient temperature at 34°C for 8 h/d (10:00-18:00). Data collection and biochemical measurements were performed every 10 d. In both age ranges, high temperature favored the innate immunity (P < 0.01) at the cost of performance (P < 0.05) during a given period, including the relative abundance of B and T-helper lymphocytes, lymphocyte proliferation ratio (B and T lymphocytes), and serum immunoglobulin contents (IgG and IgM) in the peripheral blood, as well as splenic expression of inflammation-related genes (iNOS, TLR-4, TNF-α, IL-6, and INF-γ). Compared with laying hens, growing pullets showed a time-delayed activation of immune response following heat challenge, and had no immunosuppression up to the end of exposure. Overall, the immune system of layer birds has a trade-off with production tissues in a hot environment, and exhibits distinct age-range-specific responses of acclimatization.

This experiment was conducted to investigate the effects of mulberry leaf extract (MLE) on alleviating fatty liver hemorrhagic syndrome (FLHS) in laying hens. The 576 Jing Fen laying hens of 56 weeks of age with good health and similar weights (1.76 ± 0.17 kg) were randomly divided into 6 groups, with 8 replicates in each group and 12 chickens in each replicate. The experiment lasted 56 d. The control group was fed a corn-soybean meal diet. The FLHS group was fed a high energy-low protein (HELP) diet, and the other four experimental groups were fed HELP diets supplemented with 0.04, 0.40, 0.80, and 1.20% MLE, respectively. The results showed that HELP treatment significantly induced liver injury, which indicated that the FLHS model was successfully established. MLE supplementation could alleviate the FLHS by reducing the liver index, abdominal fat percentage, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) in the serum (P < 0.05), and subsequently increase the egg production rate (P < 0.05). The laying hens fed 0.8% MLE exhibited the greatest production performance (P < 0.05) and could improve serum lipid levels. In addition, the genes associated with fatty acid synthesis (ACC, HMGR and SREBP-1C) were downregulated (P < 0.05), and genes related to fatty acid oxidation (CPT1A, AMPK, and ATGL) were found to be upregulated (P < 0.05). Supplementation with 1.2% MLE significantly reduced the relative abundance of Firmicutes and Desulfurized Bacillus (P < 0.05) and significantly increased the relative abundance of Fecal Bacillus (P < 0.05). In conclusion, MLE may regulate the mRNA expression of lipid metabolism-related genes through the AMPK signaling pathway and improve cecal microbiota balance and serum lipid levels to alleviate FLHS in laying hens and subsequently improve egg production performance.

This study was conducted to determine the influence of dietary glucosamine sulfate sodium (GSS) on laying performance, blood profiles, eggshell and inner quality of eggs and relative expression of the genes related to eggshell in laying hens at early stage. A total of 640 twenty-weeks-old Lohmann laying hens were randomly allotted to 4 treatments with 10 replicates of 16 hens each. The experiment lasted for 8 wk, and dietary treatments were: 1) CON, basal diet; 2) G1, CON + 0.2% GSS; 3) G2, CON + 0.4% GSS; 4) G3, CON + 0.6% GSS. The inclusion of GSS increased average daily feed intake, laying rate, and egg mass (P < 0.05) linearly during wk 21 to 25, 25 to 29, and 21 to 29, egg weight during wk 21 to 25 and 25 to 29, and improved (P < 0.05) feed conversion ratio linearly during wk 21 to 25. The supplementation of GSS increased (P < 0.05) albumen height quadratically, Haugh unit, calcium content, calcium mass, phosphorus content and phosphorus mass linearly at the end of 25th and 29th wk. At the end of 29th wk, the eggshell strength, eggshell weight, eggshell ratio, and eggshell thickness were increased (P < 0.05) linearly in GSS treatments compared with CON. The addition of GSS increased (P < 0.05) serum calcium, estrogen 2, and calcitonin, while decreased (P < 0.05) serum tartrate resistant acid phosphatase (TRAP), parathormone, IL-6 and prostaglandin E2 (PGE2) at the end of 29th wk. The inclusion of GSS increased (P < 0.05) the relative expression of ovocalyxin-32 and ovocalyxin-36 linearly at the end of 29th wk, and ovalbumin, osteopontin, calbindin 1, and ovocleidin-116 linearly at the end of 25th and 29th wk. Quadratic effects were observed (P < 0.05) in the laying rate during wk 21 to 25, serum TRAP and PGE2, the relative expression of ovocleidin-116 at the end of 29th wk. In summary, the inclusion of GSS up-regulated relative expression of osteopontin, ovocleidin-116, ovocalyxin-32 and ovocalyxin-36 in uterus, promoted the serum PGE2 and calcitonin, thus increased the calcium content of eggshell and finally enhanced eggshell quality.