N-Carbamylglutamate in ovo feeding improves carcass yield, muscle fiber development, and meat quality in broiler chickens.

BACKGROUND: Insufficient endogenous nutrients in the broiler embryo can lead to muscle gluconeogenesis, which ultimately affects the post-hatching performance of chicks. This study investigated the effects of in ovo feeding (IOF) of N-carbamylglutamate (NCG) on the growth hormones, carcass yield, and meat quality in broilers. Fertile eggs from a 30-week-old Ross 308 breeder flock were divided into three treatment groups: NC (non-injection), SC (100 µL saline-injection), and NCG (2 mg NCG injection). Each group had six replicates, with 70 eggs per replicate during incubation. Injections were administered on the 17.5th day of embryonic development. After hatching, 270 chicks were selected for 42-day rearing for further sampling. RESULTS: Chicks in the NCG group had significantly higher body weight (BW) and average daily gain (ADG) at the growing phase, increased growth and testosterone hormone in both feeding phases (21 and 42 days), and improved average daily gain (ADG) and food conversion ratio (FCR) in both grower and entire feeding phases (P < 0.05). Triiodothyronine (T3) and tetraiodothyronine (T4) levels, carcass yield, dressing, drum weight, breast muscle weight, drumstick weights, thighs, pectoralis major, and their part percentage of carcass were improved in the NCG group (P < 0.05), these effects were varied along feeding phases. Moreover, IOF of the NCG also improved pectoralis breast muscle color values at 24 h post mortem (P < 0.05). CONCLUSION: These results suggest that NCG injection at the late embryonic age of broiler enhances growth performance and meat quality throughout the lifespan and this can probably be attributed to an increase in thyroid and testosterone hormones, indicating potential involvement in metabolic and nutrient partitioning pathway regulation. © 2024 Society of Chemical Industry.

Improvement in growth performance and digestive function from amniotic injections of N-acetylglutamate in broiler chickens.

BACKGROUND: N-acetylglutamate (NAG) is the initial and essectial substrate in the process of de novo arginine synthesis, plays an important role in intestinal development. The aim of this study was to determine the effects of in ovo feeding of NAG, 1.5 mg/egg at 17.5 days of incubation (DOI) via amnion, on hatching performance, early intestinal histomorphometry, jejunal barrier, digestive function, and growth performance of broiler chickens between 1 and 14 days of age. RESULTS: Amniotic injection of NAG had no significant effect on hatching characteristics compared with the non-injected control group (NC group). Birds in the NAG solution-injected group (NAG group) exhibited lower average daily feed intake and better feed efficiency during a period of 1-14 days. In comparison with the NC group, the NAG group had decreased crypt depth (CD) in the ileum and increased villus height (VH) / CD in the jejunum at 7 days, and decreased CD in duodenum and significantly increased VH in the jejunum at 14 days. However, the effects of in ovo supplementation with NAG on the density of goblet cells, and gene expression of mucin 2 and alkaline phosphatase were not significant. Chicks in the NAG group had a significantly higher mRNA expression level of trypsin and maltase in jejunum at 7 days than the NC group but not at 14 days. CONCLUSION: Amniotic injections of NAG (1.5 mg/egg) at 17.5 DOI could improve early growth performance of broilers during 1-14 days after hatching by accelerating the development of the intestine and enhancing jejunal digestive function. © 2023 Society of Chemical Industry.

Effects of Pyrroloquinoline Quinone on Lipid Metabolism and Anti-Oxidative Capacity in a High-Fat-Diet Metabolic Dysfunction-Associated Fatty Liver Disease Chick Model.

Metabolic dysfunction-associated fatty liver disease (MAFLD) and its interaction with many metabolic pathways raises global public health concerns. This study aimed to determine the therapeutic effects of Pyrroloquinoline quinone (PQQ, provided by PQQ.Na2) on MAFLD in a chick model and primary chicken hepatocytes with a focus on lipid metabolism, anti-oxidative capacity, and mitochondrial biogenesis. The MAFLD chick model was established on laying hens by feeding them a high-energy low-protein (HELP) diet. Primary hepatocytes isolated from the liver of laying hens were induced for steatosis by free fatty acids (FFA) and for oxidative stress by hydrogen peroxide (H2O2). In the MAFLD chick model, the dietary supplementation of PQQ conspicuously ameliorated the negative effects of the HELP diet on liver biological functions, suppressed the progression of MAFLD mainly through enhanced lipid metabolism and protection of liver from oxidative injury. In the steatosis and oxidative stress cell models, PQQ functions in the improvement of the lipid metabolism and hepatocytes tolerance to fatty degradation and oxidative damage by enhancing mitochondrial biogenesis and then increasing the anti-oxidative activity and anti-apoptosis capacity. At both the cellular and individual levels, PQQ was demonstrated to exert protective effects of hepatocyte and liver from fat accumulation through the improvement of mitochondrial biogenesis and maintenance of redox homeostasis. The key findings of the present study provide an in-depth knowledge on the ameliorative effects of PQQ on the progression of fatty liver and its mechanism of action, thus providing a theoretical basis for the application of PQQ, as an effective nutrient, into the prevention of MAFLD.

Uterine transcriptome analysis reveals mRNA expression changes associated with the ultrastructure differences of eggshell in young and aged laying hens.

BACKGROUND: Lower eggshell quality in the late laying period leads to economic loss. It is a major threat to the quality and safety of egg products. Age-related variations in ultrastructure were thought to induce this deterioration. Eggshell formation is a highly complex process under precise regulation of genes and biological pathways in uterus of laying hens. Herein, we evaluated the physical, mechanical and ultrastructure properties of eggshell and conducted RNA sequencing to learn the transcriptomic differences in uterus between laying hens in the peak (young hens) and late phase (aged hens) of production. RESULTS: The declined breaking strength and fracture toughness of eggshell were observed in aged hen group compared to those in young hen group, accompanied with ultrastructure variations including the increased thickness of mammillary layer and the decreased incidence of early fusion. During the initial stage of eggshell formation, a total of 183 differentially expressed genes (DEGs; 125 upregulated and 58 downregulated) were identified in uterus of laying hens in the late phase in relative to those at peak production. The DEGs annotated to Gene Ontology terms related to antigen processing and presentation were downregulated in aged hens compared to young hens. The contents of proinflammatory cytokine IL-1ß in uterus were higher in aged hens relative to those in young hens. Besides, the genes of some matrix proteins potentially involved in eggshell mineralization, such as ovalbumin, versican and glypican 3, were also differentially expressed between two groups. CONCLUSIONS: Altered gene expression of matrix proteins along with the compromised immune function in uterus of laying hens in the late phase of production may conduce to age-related impairments of eggshell ultrastructure and mechanical properties. The current study enhances our understanding of the age-related deteriorations in eggshell ultrastructure and provides potential targets for improvement of eggshell quality in the late laying period.

Effect of in ovo feeding of ß-hydroxy-ß-methylbutyrate on hatchability, muscle growth and performance in prenatal and posthatch broilers.

BACKGROUND: ß-Hydroxy-ß-methylbutyrate (HMB) is the metabolite of leucine that plays an important role in muscle protein metabolism. The objective of the present study was to determine the effects of in ovo feeding (IOF) of HMB at 7 days of incubation (DOI) via air cell or 18 DOI via amnion on hatchability, muscle growth and performance in prenatal and posthatch broilers. RESULTS: IOF of HMB via air cell at 7 DOI increased hatchability by 4.34% compared with the control (89.67% versus 85.33%). Birds in IOF groups exhibited higher body weight, average daily body weight gain and pectoral muscle percentage. Furthermore, IOF of HMB significantly increased the level of plasma growth hormone, insulin and insulin-like growth factor-1. Chicks hatched from IOF treatment had larger diameters of muscle fiber and higher mitotic activity of satellite cells at early posthatch age. IOF of HMB activated satellite cells by upregulation of mRNA expression of myogenic transcription factors, myogenic differentiation one (MyoD) and myogenin. Chicks hatched from air cell injection group had higher pectoral muscle percentage at 5 d posthatch and greater satellite cell mitotic activity at 7 d posthatch than counterparts from amnion injection group. CONCLUSIONS: IOF of HMB via amnion at 18 DOI or especially via air cell at 7 DOI could be used as an effective approach to enhance hatchability, productive performance and breast muscle yield in broilers. © 2019 Society of Chemical Industry.

Growth performance, carcass characteristics, meat quality and serum profile of broiler chicks fed on housefly maggot meal as a replacement of soybean meal.

A study was conducted to invesstigate the housefly maggot meal (HMM) as an alternative protein source to replace the soybean meal in broiler chick's diet. A total of 720 1-day-old male broiler chicks were divided into three groups and fed diets formulated with HMM to replace soybean meal at the rate of 0%, 4% and 8%. The study lasted for 42 days in two phases. Results showed that HMM addition did not markedly affect body weight, average daily body weight gain and average daily feed intake of the broiler chicks. Feed conversion ratio increased linearly (1-21 days) in starter or quadratically (22-42 days) in the grower phase. HMM non-significantly increased the feed intake and body weight during the grower phase. Slight changes were observed for decrease of blood biochemical indices in the platelets (day 21), and alkaline phosphatase and lysozyme (day 42), and increase for red blood cells, packed cell volume, total protein and uric acid on day 42; however, the fluctuations were within the physiological range. Non-significant effects were observed for carcass composition and meat quality, except that HMM numerically reduced the shear force of breast muscle (linear, p = .058). These results are the strong evidence that HMM can be used as an alternative protein source at 8% in broiler chick's diet without any adverse effect on chick's performance.

Transcriptome analysis reveals mechanism underlying the differential intestinal functionality of laying hens in the late phase and peak phase of production.

BACKGROUND: The compromised performance of laying hens in the late phase of production relative to the peak production was thought to be associated with the impairment of intestinal functionality, which plays essential roles in contributing to their overall health and production performance. In the present study, RNA sequencing was used to investigate differences in the expression profile of intestinal functionality-related genes and associated pathways between laying hens in the late phase and peak phase of production. RESULTS: A total of 104 upregulated genes with 190 downregulated genes were identified in the ileum (the distal small intestine) of laying hens in the late phase of production compared to those at peak production. These upregulated genes were found to be enriched in little KEGG pathway, however, the downregulated genes were enriched in the pathways of PPAR signaling pathway, oxidative phosphorylation and glutathione metabolism. Besides, these downregulated genes were mapped to several GO clusters in relation to lipid metabolism, electron transport of respiratory chain, and oxidation resistance. Similarly, there were lower activities of total superoxide dismutase, glutathione S-transferase and Na+/K+-ATPase, and reductions of total antioxidant capacity and ATP level, along with an elevation in malondialdehyde content in the ileum of laying hens in the late phase of production as compared with those at peak production. CONCLUSIONS: The intestine of laying hens in the late phase of production were predominantly characterized by a disorder of lipid metabolism, concurrent with impairments of energy production and antioxidant property. This study uncovers the mechanism underlying differences between the intestinal functionality of laying hens in the late phase and peak phase of production, thereby providing potential targets for the genetic control or dietary modulation of intestinal hypofunction of laying hens in the late phase of production.

Effect of dietary protein sources on production performance, egg quality, and plasma parameters of laying hens.

OBJECTIVE: This study was conducted to evaluate the effects of dietary protein sources (soybean meal, SBM; low-gossypol cottonseed meal, LCSM; double-zero rapeseed meal, DRM) on laying performance, egg quality, and plasma parameters of laying hens. METHODS: A total of 432 32-wk-old laying hens were randomly divided into 6 treatments with 6 replicates of 12 birds each. The birds were fed diets containing SBM, LCSM100, or DRM100 individually or in combination with an equal amount of crude protein (CP) (LCSM50, DRM50, and LCSM50-DRM50). The experimental diets, which were isocaloric (metabolizable energy, 11.11 MJ/kg) and isonitrogenous (CP, 16.5%), had similar digestible amino acid profile. The feeding trial lasted 12 weeks. RESULTS: The daily egg mass was decreased in the LCSM100 and LCSM50-DRM50 groups (p<0.05) in weeks 41 to 44. The LCSM50 group did not affect egg production compared to the SBM group in weeks 41 to 44 (p>0.05) and showed increased yolk color at the end of the trial (p<0.05). Compared to the SBM group, the LCSM100 and LCSM50-DRM50 groups showed decreased albumen weight (p<0.05), CP weight in the albumen (p<0.05) and CP weight in the whole egg (p<0.05) at 44 weeks. Plasma total protein (TP) levels were lower in the LCSM100 group than in the SBM group at 44 weeks (p<0.05); however, TP, albumin, and globulin levels were not significantly different between the LCSM50 group and the SBM group or between the DRM50 group and the SBM group (p>0.05). CONCLUSION: Together, our results suggest that the LCSM100 or DRM100 diets may produce the adverse effects on laying performance and egg quality after feeding for 8 more weeks. The 100.0 g/kg LCSM diet or the148.7 g/kg DRM diet has no adverse effects on laying performance and egg quality.

The change of albumen quality during the laying cycle and its potential physiological and molecular basis of laying hens.

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.

Effect of Genistein and Glycitein on production performance, egg quality, antioxidant function, reproductive hormones and related-genes in pre-peak laying hens.

Genistein (GEN) and Glycitein (GLY), are types of isoflavone extracted mainly from soy plants, although GEN is associated with stronger antioxidant and growth-promoting effects. The impact of dietary GEN and GLY on reproductive performance, egg quality, and bone quality were investigated in the study. Additionally, to explore the underlying mechanism of action, the serum hormone levels and reproductive-related genes were investigated. A total of 378 Hy-Line Brown laying hens (120 days old) were randomly allocated to 3 dietary groups (Control), (GLY, and GEN at 50 mg/kg respectively) for a period of 8 wk. Each treatment has 126 birds (7 replicates of 18 birds each). Results were analyzed in 2 phases: wk 1 to 4, and 5 to 8 of feeding trial. The results indicated that supplemental GEN significantly increased egg number, hen-day production (HDP), and egg mass during wk 1 to 4, whereas, both glycitein and genistein increased egg number, egg weight, egg mass, HDP and improved feed-egg-ratio during wk 5 to 8. Egg quality analysis revealed significant improvements in eggshell quality; gloss, thickness, strength, and albumen quality indices (albumen height, Haugh unit, thick albumen fraction) due to dietary treatments. Also, the tibia strength, Ca content in the tibia ash and bone mineral content, were significantly increased by the dietary treatments. Significant increases in the serum levels of E2, LH, FSH, T3, T4, and GH, and the activity of antioxidant enzymes; SOD, CAT, GSH while reducing the level of MDA, was notable with the treatments. Additionally, reproductive-related genes: ESR1, FSHR, PRLR, GNRH1 were significantly upregulated by the supplementation of GEN and GLY. The efficacy of GEN in relation to the evaluated parameters was superior to that of GLY. Conclusively, we speculate that the improvement on laying performance, egg quality and tibia quality may be related to promoting effect of isoflavones on calcium metabolism, antioxidant function, reproductive hormones and related genes. Therefore, supplemental GEN at a dosage level of 50 mg/kg, can be used to promote laying performance, sustain egg production and maintain the physiological function of young laying hens.

Fermented soybean meal improved laying performance and egg quality of laying hens by modulating cecal microbiota, nutrient digestibility, intestinal health, antioxidant and immunological functions.

Antinutritional factors in feedstuffs may limit their utilization in livestock production, but fermentation process can be used to improve feed quality; however, studies on fermented soybeans for laying hens remain limited. We investigated the effect of fermented soybean meal (FSBM) at various inclusion levels as a partial replacement for soybean meal (SBM) on egg production, egg quality, amino acid digestibility, gut morphology and microbiota, antioxidant capacity and immune response of young laying hens. A total of 360 Hy-line Brown laying hens aged 18 weeks were selected and divided into 5 groups of 6 replicates each and 12 birds per replicate. The control group received a basal diet while the trial group received the basal diet with FSBM included at 2.5%, 5.0%, 7.5% and 10.0%, respectively, for 12 weeks. Our findings revealed that the nutritional value of FSBM was higher compared to that of SBM in terms of reduced content of trypsin inhibitors and increased contents of crude protein, amino acids and minerals. FSBM enhanced egg production (P < 0.05), feed-to-egg ratio (P < 0.05), and albumen quality (albumen height and Haugh unit) (P < 0.05). Furthermore, FSBM improved apparent fecal amino acid digestibility (P < 0.05), gut morphology (increased villus height, villus width, villus height-to-crypt depth ratio and decreased crypt depth) (P < 0.05), antioxidant capacity (reduced malondialdehyde and increased catalase, total superoxide dismutase, glutathione peroxidase and total antioxidant capacity) (P < 0.05) and immune function (increased concentrations of IgG, IgA, and IgM; increased levels of transforming growth factor beta and Toll-like receptor 2; and reduced levels of interleukin 1ß and tumor necrosis factor alpha) (P < 0.05). Further analysis showed that FSBM altered the composition of the gut microbiota favoring beneficial microbes. These findings suggest that probiotic fermentation improved the nutritional value of SBM. The inclusion of FSBM in the diets of laying hens at 2.5% or 5.0% improved amino acid digestibility, gut health, immune function, egg production and egg quality.

Dietary supplementation with calcitriol or quercetin improved eggshell and bone quality by modulating calcium metabolism.

This study was aimed to investigate the effects of dietary calcitriol or quercetin supplementation on eggshell and bone quality of laying hens. In trial 1, 72 Hy-Line Brown layers (80-week-old) with weak-shelled strength (25 to 30 N) were assigned into 4 dietary treatments with 6 replicates of 3 birds and fed a basal diet (4% calcium level) or basal diets supplemented with 0.5% calcium, 5 µg/kg calcitriol or 500 mg/kg quercetin for 4 weeks. In trial 2, 360 Hy-Line Brown layers (60-week-old) were divided into 3 groups with 8 replicates of 15 birds: control group (basal diet), calcitriol group (basal diet + 5 µg/kg calcitriol), and quercetin group (basal diet + 500 mg/kg quercetin). This trial lasted for 12 weeks. The results showed that dietary calcitriol or quercetin improved eggshell quality in both trials (P < 0.05). In trial 2, compared with the control group, both calcitriol and quercetin supplementations improved femoral bone quality, calcium retention of hens and calcium content in uterine fluid at 18.5 h post-oviposition (PO) (P < 0.05), along with enhancing uterine morphology. Compared to the control group, supplemental calcitriol or quercetin up-regulated the relative mRNA expression levels of uterine transient receptor potential cation channel, subfamily V, member 6 (TRPV6) at 8.5 h PO and plasma membrane calcium-ATPase (PMCA), vitamin D receptor (VDR), estrogen receptor alpha (ERα) at 18.5 h PO (P < 0.05), but down-regulated the uterine caspase 3 (CASP3) relative mRNA expression level at 8.5 h PO (P < 0.05). Meanwhile, the femoral relative mRNA expression levels of tartrate-resistant acid phosphatase (TRAP) (up-regulated at 8.5 and 18.5 h PO) and alkaline phosphatase (ALP) (up-regulated at 8.5 h PO but down-regulated at 18.5 h PO) were also affected by calcitriol or quercetin supplementation (P < 0.05). Compared to the calcitriol, quercetin increased hen-day egg production and femoral medullary bone volume/bone tissue volume but reduced femoral stiffness (P < 0.05), which were accompanied by increased relative mRNA expression levels of uterine TRPV6, estrogen receptor beta (ERß) at 18.5 h PO (P < 0.05). Overall, both dietary calcitriol and quercetin could improve eggshell and bone quality by modulating calcium metabolism of aged layers. Compared to calcitriol, dietary quercetin up-regulated the expression of uterine calcium transporters, without affecting eggshell quality.

Inclusion of guanidinoacetic acid in a low metabolizable energy diet improves broilers growth performance by elevating energy utilization efficiency through modulation serum metabolite profile.

This study was aimed to explore the elevating energy utilization efficiency mechanism for the potentially ameliorative effect of guanidinoacetic acid (GAA) addition on growth performance of broilers fed a low metabolizable energy (LME) diet. A total of 576 d old broilers were randomly allocated to one of the six treatments: a basal diet (normal ME, positive control, PC), or an LME diet (50 kcal/kg reduction in ME, negative control, NC) supplemented with 0.02%, 0.04%, 0.06%, and 0.08% GAA from 1 to 42 d of age, respectively. The GAA fortification in LME diet linearly or quadratically dropped (P < 0.05) the feed conversion ratio (FCR) from 22 to 42 and 1 to 42 d of age, abdominal fat rate on day 42, serum alanine aminotransferase (ALT) on day 21, and serum creatinine (CREAN) on days 21 and 42, elevated (P < 0.05) breast muscle rate and leg muscle rate on day 42, serum creatine kinase (CK) on days 21 and 42, as well as alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) on day 21. The dietary optimal GAA levels were 0.03%-0.08% based on the best-fitted quadratic models (P < 0.03) of the above parameters. Thus, the PC, LME, and 0.04% GAA-LME groups were selected for further analysis. Serum essential amino acids (EAA) tryptophan, histidine and arginine, non-essential amino acids (NEEA) serine, glutamine and aspartic acid were significantly decreased (P < 0.05), compared to PC diet by LME or 0.04% GAA-LME diet. 0.04% GAA-LME group reversed (P < 0.05) the reduction of arginine, 3-methyhistidine, and 1-methylhistidine by LME diet. Besides, six birds at 28 d of age from LME and 0.04% GAA-LME groups were selected for energy utilization observation in calorimetry chambers. The results demonstrated that 0.04% GAA-LME group significantly improved (P < 0.05) the ME intake (MEI) and net energy (NE) compared to the LME diet. Overall, these findings suggest that 0.04% GAA is the ideal dose of broilers fed the LME diet, which can significantly improve the growth performance and carcass characteristics by modulation of creatine metabolism through elevating serum CK activity and arginine concentration.

Guanidinoacetic acid (GAA) has been found to elevate energy utilization efficiency in broilers; however, the underlying mechanisms remain unclear. We investigated the effects of GAA addition in low metabolizable energy (LME) diet on growth performance, carcass characteristics and serum biochemical indices of broilers, and found that GAA addition linearly or quadratically dropped the feed conversion ratio from 22 to 42 and 1 to 42 d of age, abdominal fat rate on day 42, serum alanine aminotransferase on day 21, and serum creatinine on days 21 and 42, elevated breast muscle and leg muscle rate on day 42, serum creatine kinase, alkaline phosphatase, as well as lactate dehydrogenase on days 21 or 22. The dietary optimal GAA levels were 0.03%-0.08% based on the best-fitted quadratic models of the above parameters. Thus, further analysis was conducted and found that 0.04% GAA reversed the reduction of arginine, 3-methyhistidine, and 1-methylhistidine and improved the ME intake and net energy compared to the LME diet. These findings suggested that 0.04% GAA is the ideal dose for enhancing the energy utilization of broilers fed the LME diet, GAA addition can significantly improve the growth performance by elevating energy utilization efficiency through modulation serum metabolite profile.

Decreased eggshell strength caused by impairment of uterine calcium transport coincide with higher bone minerals and quality in aged laying hens.

BACKGROUND: Deteriorations in eggshell and bone quality are major challenges in aged laying hens. This study compared the differences of eggshell quality, bone parameters and their correlations as well as uterine physiological characteristics and the bone remodeling processes of hens laying eggs of different eggshell breaking strength to explore the mechanism of eggshell and bone quality reduction and their interaction. A total of 240 74-week-old Hy-line Brown laying hens were selected and allocated to a high (HBS, 44.83 ± 1.31 N) or low (LBS, 24.43 ± 0.57 N) eggshell breaking strength group. RESULTS: A decreased thickness, weight and weight ratio of eggshells were observed in the LBS, accompanied with ultrastructural deterioration and total Ca reduction. Bone quality was negatively correlated with eggshell quality, marked with enhanced structures and increased components in the LBS. In the LBS, the mammillary knobs and effective layer grew slowly. At the initiation stage of eggshell calcification, a total of 130 differentially expressed genes (DEGs, 122 upregulated and 8 downregulated) were identified in the uterus of hens in the LBS relative to those in the HBS. These DEGs were relevant to apoptosis due to the cellular Ca overload. Higher values of p62 protein level, caspase-8 activity, Bax protein expression and lower values of Bcl protein expression and Bcl/Bax ratio were seen in the LBS. TUNEL assay and hematoxylin-eosin staining showed a significant increase in TUNEL-positive cells and tissue damages in the uterus of the LBS. Although few DEGs were identified at the growth stage, similar uterine tissue damages were also observed in the LBS. The expressions of runt-related transcription factor 2 and osteocalcin were upregulated in humeri of the LBS. Enlarged diameter and more structural damages of endocortical bones and decreased ash were observed in femurs of the HBS. CONCLUSION: The lower eggshell breaking strength may be attributed to a declined Ca transport due to uterine tissue damages, which could affect eggshell calcification and lead to a weak ultrastructure. Impaired uterine Ca transport may result in reduced femoral bone resorption and increased humeral bone formation to maintain a higher mineral and bone quality in the LBS.

Dietary 25-Hydroxycholecalciferol Supplementation as a Vitamin D3 Substitute Improves Performance, Egg Quality, Blood Indexes, Jejunal Morphology, and Tibia Quality in Late-Phase Laying Hens.

This study aimed to investigate whether a dietary 25-OHD3 addition improved the performance, egg quality, blood indexes, antioxidant status, jejunal morphology, and tibia quality of aged laying hens compared to a dietary VD3 addition. A total of 270 Hy-Line Brown laying hens at 55 wk of age were randomly assigned to three dietary treatments with six replicates (15 birds per replicate with 3 birds per cage). Chickens were fed a corn-soybean meal diet supplementation of 4000 IU/kg VD3 (control group), 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 (experimental group 1), or 50 µg/kg 25-OHD3 and 4000 IU/kg VD3 (experimental group 2) for 12 weeks. The results demonstrated that 25-OHD3 caused a significant increase in the laying rate, especially in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group; the laying rate reached the maximum compared with other groups after 12 weeks (p < 0.05). However, there were no significant effects on the average egg weight, average daily feed intake, or feed-to-egg ratio (p > 0.05). A dietary supplementation of 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 provided an improved eggshell strength, thick albumen height, and Haugh unit after 12 weeks (p < 0.05). Further analysis of the blood indexes showed that alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, calcium, and phosphorus were enhanced significantly in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group, while the content of total bilirubin decreased significantly (p < 0.05). In addition, the 25-OHD3 addition in diets improved the calcium and phosphorus contents in the serum (p < 0.05). The concentrations of 25-OHD3, parathyroid hormones, follicle-stimulating hormone, and progesterone were increased in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group, and the levels of cortisol, calcitonin, bone gla protein, and endotoxin in the serum reached a minimum in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 group (p < 0.05), which constitutes an advantage for the aged laying hens. The antioxidant enzyme activities and free radical scavenging abilities in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group increased markedly, and the MDA level decreased significantly in the 50 µg/kg 25-OHD3 + 4000 IU/kg VD3 group (p < 0.05). Improvements in jejunal morphology and intestinal integrity resulted in an increased villi-length-to-crypt-depth ratio in the 50 µg/kg 25-OHD3 + 2000 IU/kg VD3 group (p < 0.05). Dietary 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 additions improved the tibia quality, including fresh tibia weight, strength, mineral content (Ca), and trabeculae area (p < 0.05). Taken together, compared with the dietary VD3 addition, dietary supplementation of 25-OHD3 supported a stable physiological status for sustained egg production, egg quality, and bone quality in late-phase laying hens, and the addition levels of 50 µg/kg 25-OHD3 and 2000 IU/kg VD3 had the best effect. Therefore, this could provide a theoretical basis for the use of 25-OHD3 as a substitute forVD3.

Histological and molecular difference in albumen quality between post-adolescent hens and aged hens.

The decline in albumen quality resulting from aging hens poses a threat to the financial benefits of the egg industry. Exploring the underlying mechanisms from the perspective of cell molecules of albumen formation is significant for the efficient regulation of albumen quality. Two individual groups of Hy-Line Brown layers with ages of 40 (W40) and 100 (W100) wk old were used in the present study. Each group contained over 2,000 birds. This study assessed the egg quality, biochemical indicators and physiological status of hens between W40 and W100. Subsequently, a quantitative proteomic analysis was conducted to identify differences in protein abundance in magnum tissues between W40 and W100. In the W40 group, significant increases (P < 0.05) were notable for albumen quality (thick albumen solid content, albumen height, Haugh unit), serum indices (calcium, estrogen, and progesterone levels), magnum histomorphology (myosin light-chain kinase content, secretory capacity, mucosal fold, goblet cell count and proportion) as well as the total antioxidant capacity of the liver. However, the luminal diameter of the magnum, albumen gel properties and random coil of the albumen were increased (P < 0.05) in the W100 group. The activity of glutathione, superoxidase dismutase, and malondialdehyde in the liver, magnum, and serum did not vary (P > 0.05) among the groups. Proteomic analysis revealed the identification of 118 differentially expressed proteins between the groups, which comprised proteins associated with protein secretion, DNA damage and repair, cell proliferation, growth, antioxidants, and apoptosis. Furthermore, Kyoto Encyclopedia of Genes pathway analysis revealed that BRCA2 and FBN1 were significantly downregulated in Fanconi anemia (FA) and TGF-ß signaling pathways in W100, validated through quantitative real-time PCR (qRT-PCR). In conclusion, significant age-related variations in albumen quality, and magnum morphology are regulated by proteins involved in antioxidant capacity.

TMT-based quantitative proteomic analysis reveals eggshell matrix protein changes correlated with eggshell quality in Jing Tint 6 laying hens of different ages.

The decline in eggshell quality resulting from aging hens poses a threat to the financial benefits of the egg industry. The deterioration of eggshell quality with age can be attributed to changes in its ultrastructure and chemical composition. Specific matrix proteins in eggshells have a role in controlling crystal growth and regulating structural organization. However, the variations in ultrastructure and organic matrix of eggshells in aging hens remain poorly understood. This study assessed the physical traits, mechanical quality, chemical content, as well as the microstructural and nanostructural properties of eggs from Jing Tint 6 hens at 38, 58, 78, and 108 wk of age. Subsequently, a quantitative proteomic analysis was conducted to identify differences in protein abundance in eggshells between the ages of 38 and 108 wk. The results indicated a notable decline in shell thickness, breaking strength, index, fracture toughness, and stiffness in the 108-wk-age group compared to the other groups (P < 0.05). The ultrastructure variations primarily involved an increased ratio of the mammillary layer and a reduced thickness of the effective layer of eggshell in the 108-wk-age group (P < 0.05). However, no significant differences in eggshell compositions were observed among the various age groups (P > 0.05). Proteomic analysis revealed the identification of 76 differentially expressed proteins (DEPs) in the eggshells of the 38-wk-age group and 108-wk-age group, which comprised proteins associated with biomineralization, calcium ion binding, immunity, as well as protein synthesis and folding. The downregulation of ovocleidin-116, osteopontin, and calcium-ion-related proteins, together with the upregulation of ovalbumin, lysozyme C, and antimicrobial proteins, has the potential to influence the structural organization of the eggshell. Therefore, the deterioration of eggshell quality with age may be attributed to the alterations in ultrastructure and the abundance of matrix proteins.

Uterine microbial communities and their potential role in the regulation of epithelium cell cycle and apoptosis in aged hens.

BACKGROUND: Alterations of the uterine microbiome are closely associated with various intrauterine diseases and physiological conditions, which are well-established in mammals. However, as representative oviparous animals, the research on the uterine microbial ecosystem and its functions with physiological homeostasis is limited in chickens. Additionally, continuous egg-laying disrupts the oviducal immune defenses of aged hens, susceptible to pathogen invasion, causing poor egg quality and food-borne infections in humans. Here, we investigated aging-related changes in the oviduct microbial colonization and transmission from the gut to eggs and their roles in a hen model. RESULTS: The results of 16S rDNA sequencing showed significant differences in the oviduct microbial composition between young (38 weeks) and aged (77 weeks) laying hens. SourceTracker analysis further revealed differences in the effects of microbial transmission on the oviducal microbiota between young and aged hens. Enhanced barrier defense with cell apoptosis suppression and cell cycle arrest of the uterus were observed in aged hens reducing microbial transmission from the lower to upper reproductive tract. In addition, a total of 361 significantly differential metabolites were identified using metabolomics in the aged uterine microbiota, especially in products of amino acid metabolism and biosynthesis of various secondary metabolites, which might have essential effects on cell apoptosis by regulating immune responses and cell cycle. Notably, antibiotics disrupted uterine microbiota by dietary intervention and direct perfusion did not retard aging-related physiological changes but further aggravated aging processes by disrupting the cell cycle and apoptosis. CONCLUSIONS: The microbiota continuum along the reproductive tract in aged birds differs from that in young birds, especially with a significant shift in the uterus. The aged uterine microbiota probably contributes to the regulation of cell cycle and apoptosis by microbial metabolites primarily involved in amino acid metabolism and biosynthesis of various secondary metabolites. These findings provide new insights into the roles of the reproductive tract microbiota in regulating the cell programming of the aged host, contributing to the exploration of the microbiome as a target for diagnosing aging health status and therapy for gynecological diseases in women. Video Abstract.

Effects of Dietary Rare Earth Chitosan Chelate on Performance, Egg Quality, Immune and Antioxidant Capacity, and Intestinal Digestive Enzyme Activity of Laying Hens.

Rare earth chitosan chelate salt (RECC) is a potential feed additive and is a product of the chelation effect between rare earth ions and chitosan. This research study aims to explore the effects of dietary RECC on performance, egg quality, intestinal digestive function, and the immune and antioxidant capacity of laying hens in the late phase of production. A total of 360 56-week-old Dawu Jinfeng laying hens were randomly allotted into four treatment groups with six replicates per treatment and 15 birds per replicate. The laying hens were fed the basal diet supplemented with, respectively, 0 (control: CON), 100 (R1), 200 (R2), and 400 (R3) mg/kg for 8 weeks. Dietary RECC significantly improved average daily feed intake (ADFI) and average daily egg yield in both linear and quadratic manner (p < 0.05). In addition, albumen height and HU were improved significantly (p < 0.05) in a dose-dependent manner of RECC. In addition, a significant decrease (p < 0.05) in serum TP, IgA, and MDA for the R1 group and IgG in the R2 group were notable, while the increase in serum TP and decrease in T-AOC were found for R3 dietary group compared to CON (p < 0.05). The level of intestinal IL-2 and TNF-α was decreased by dietary RECC (p < 0.01). The activities of the digestive enzyme (α-Amylase, lipase, and Trypsin) showed a quadratic change with an increase and then decrease in response to increasing dose of RECC, 200 mg/kg RECC significantly increased the activity of lipase and Trypsin (p < 0.01). Supplementation of dietary RECC at low doses compared to higher doses impacted positive effects on the antioxidant capacity and immune function (p < 0.05). The utilization of RECC as a feed additive in the diet of aged laying hens exerted beneficial effects on egg production, albumen quality, humoral immunity, inflammatory response, and activity of digestive enzymes. Thus, the regulation of antioxidant capacity and duodenal function via increased enzyme activity and immune and inflammatory response were critical to the improvement of laying performance and egg quality in aged hens. The optimal supplemental dose is 100-200 mg/kg.

CDCA7 serves as a novel prognostic marker in human hepatocellular carcinoma.

c-Myc oncogene plays an important role in tumorigenesis, cell division cycle associated 7 (CDCA7), recently found that it is a direct target gene of c-Myc, is upregulated in many tumors, but its role in tumor progression is still poorly understood. CDCA7 expression and prognosis were analyzed in hepatocellular carcinoma using TIMER2.0 and Kaplan-Meier databases, while genomic changes were studied using cbioportal. LinkedOmics identified relevant genes and WebGestalt analyzed the associated pathways. Protein interaction networks were explored using the STRING database, and the core PPI network was analyzed with the MCODE plugin of Cytoscape. CDCA7 expression was detected in 30 paired HCC specimens by real-time PCR, and its effect on HCC cell proliferation was determined in vitro. CDCA7 expression was frequently up-regulated in human hepatocellular carcinoma (HCC), and its expression was positively correlated with prognosis. The TIMER2.0 database showed that CDCA7 was differentially expressed in hepatocellular carcinoma, with high expression in tumor tissues and low expression in normal tissues. The Kaplan-Meier database shows that high CDCA7 expression has a worse prognosis. The cBioportal database showed that the genomic change rate of CDCA7 in hepatocellular carcinoma was 2.15%, including mutations, amplifications, and deep deletions. Pathway analysis of related genes showed that CDCA7-related genes were mainly focused on cell division-related pathways. The experimental results also validate our study. CDCA7 could contribute to HCC progression and raise the possibility that CDCA7 is a potential new therapeutic target for HCC treatment.