Carotenoid content and deposition efficiency in yolks of laying hens fed with dent corn hybrids differing in grain hardness and processing.

Yolk carotenoid profile reflects the hen diet when corn grain is the only source of carotenoids, but corn origin and processing may affect carotenoid utilization. In the present study, 2 commercial dent corn hybrids differing in grain hardness (soft- and hard-type) were dried at low (40°C) and high (85°C) temperature and ground through a 5- and 9-mm sieve to investigate their effects on carotenoid bioavailability in laying hens. With 3 hens per cage, 168 Lohmann Brown laying hens were allocated to 8 dietary treatments (2 hybrids × 2 drying temperatures × 2 grinding sieves) in a completely randomized design (8 treatments × 7 cages). The trial lasted 8 wk, during which eggs were collected for analysis every 3 d until carotenoid content stabilized, and then once a week until the end of the experiment. The carotenoid profile of the experimental diets and yolks was analyzed using an HPLC method and deposition efficiency was calculated based on carotenoid contents, yolk weight, egg production and diet intake. The deposition efficiency for lutein, zeaxanthin, α- and ß-cryptoxanthin, and ß-carotene averaged 27.37, 18.67, 6.29, 3,32, and 0.94%, respectively. As expected, the tested hybrids highly affected the carotenoid content in egg yolk due to their differences in carotenoid profile. Interestingly, hard- and soft-type hybrids differed in the deposition efficiency for all individual carotenoids but not for the total carotenoids. High grain drying temperature tended to increase the bioavailability of lutein and zeaxanthin in both hybrids. For the hard-type hybrid, the content of ß-carotene in egg yolk was higher when grains were dried at a high temperature, while the opposite response was found in the soft-type hybrid. The effect of grinding sieve size was important for the zeaxanthin bioavailability in the soft-type hybrid only. In conclusion, our findings showed that corn hybrid had a primary influence on the carotenoid content in the yolks of laying hens, but grain processing may change the bioavailability of carotenoids.

Mulberry branch fiber improved lipid metabolism and egg yolk fatty acid composition of laying hens via the enterohepatic axis.

BACKGROUND: The utilization of mulberry branch fiber (MF), the largest by-product of the sericulture industry, is an important issue. Supplementation with MF as a dietary fiber for poultry may serve as a useful application. However, little is known about the effects of MF on liver lipid metabolism and egg yolk fatty acid composition of laying hens and their underlying mechanisms. In this study, we performed a multi-omics investigation to explore the variations in liver lipid metabolism, egg yolk fatty acid composition, gut microbiota, and the associations among them induced by dietary MF in laying hens. RESULTS: Dietary MF had no harmful effects on the laying performance or egg quality in laying hens. The enzyme activities associated with lipid metabolism in the liver were altered by the addition of 5% MF, resulting in reduced liver fat accumulation. Furthermore, dietary 5% MF induced the variation in the fatty acid profiles of egg yolk, and increased the polyunsaturated fatty acid (PUFA) content. We observed a significant reduction in the diversity of both gut bacteria and changes in their compositions after the addition of MF. Dietary MF significantly increased the abundance of genes involved in fatty acid biodegradation, and short-chain fatty acids biosynthesis in the gut microbiota of laying hens. The significant correlations were observed between the liver lipid metabolism enzyme activities of hepatic lipase, lipoprotein lipase, and total esterase with gut microbiota, including negative correlations with gut microbiota diversity, and multiple correlations with gut bacteria and viruses. Moreover, various correlations between the contents of PUFAs and monounsaturated fatty acids in egg yolk with the gut microbiota were obtained. Based on partial-least-squares path modeling integrated with the multi-omics datasets, we deduced the direct effects of liver enzyme activities and gut bacterial compositions on liver fat content and the roles of liver enzyme activities and gut bacterial diversity on egg yolk fatty acid composition. CONCLUSIONS: The results indicate that dietary MF is beneficial to laying hens as it reduces the liver fat and improves egg yolk fatty acid composition through the enterohepatic axis. Video Abstract.

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.

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.

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.

Research Note: Effects of the intermittent feeding of microencapsulation essential oil on laying performance, egg quality, immune response, intestinal morphology, and oxidation status of laying hens.

The aim of this study was to evaluate the effect of microencapsulated essential oils (MEO) on the laying performance, egg quality, immunity, intestinal morphology, and oxidative status of laying hens. A total of 640 Hy-line Brown laying hens, 41 wk of age, were randomly divided into 4 groups, each with 8 replicates containing 20 birds per replicate. The dietary conditions tested included a basal diet (Control) or the basal diet supplemented with various levels of MEO at 100 mg/kg (MEO100), 300 mg/kg (MEO300), and 500 mg/kg (MEO500). The three treatment groups were intermittently fed MEO, following an alternating schedule of 1 wk on and 1 wk off for a total of 56 d. Results showed that feeding MEO at levels of 300 and 500 mg/kg improved both egg production and feed conversion ratios compared to the control group. Hens consumed MEO-supplemented diets exhibited a significant decrease in the breaking egg ratio (P < 0.05) compared to those fed the control diet. Shell thickness and Haugh unit values significantly increased in the groups receiving 300 and 500 mg/kg of MEO (P < 0.05). Both the MEO300 and MEO500 treatments led to improvements in immunoglobulin (IgA, IgM, and IgG) and cytokine (IL-2 and IFN-γ) levels in serum. Hens in the MEO300 and MEO500 groups exhibited higher values for parameters related to intestinal morphometry compared to the control group. Furthermore, supplementation with 300 and 500 mg/kg of MEO enhanced the antioxidant capacity of plasma, as evidenced by increased activities of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), and catalase (CAT) (P < 0.05). In summary, the intermittent feeding of MEO improved egg production, enhanced antioxidative processes, immune functions, and intestinal morphology, leading to an amelioration in the egg quality of laying hens. Our data demonstrate that supplementation of 300 mg/kg of MEO in feed can significantly improve animal health and egg quality. Implementation of these feeding practices could have a positive economic impact on poultry and egg industry.

ß-Hydroxy-ß-methylbutyrate: A feed supplement influencing performance, bone metabolism, intestinal morphology, and muscle quality of laying hens: a preliminary one-point study.

Laying hens, selectively bred for high egg production, often suffer from bone fragility and fractures, impacting their welfare and causing economic losses. Additionally, gut health and muscle quality are crucial for overall health and productivity. This study aimed to evaluate the effects of ß-Hydroxy-ß-methylbutyrate (HMB) supplementation on performance, bone metabolism, intestinal morphology, and muscle quality in laying hens. Forty-eight Bovans Brown hens were divided into a control group and an HMB-supplemented group (0.02% HMB in diet). The study spanned from the 31st to the 60th wk of age. Assessments included bone mechanical testing, serum hormonal analysis, histological analysis of bone and intestine, and muscle quality analysis. The HMB supplementation led to decreased feed intake without affecting body weight or laying rate in laying hens. It caused an increase in both mean daily and total egg weight, indicating improved feed utilization, without influencing the feed intake to egg weight ratio. Enhanced bone formation markers and altered intestinal morphometric parameters were observed, along with improved trabecular bone structure. However, no changes in measured other bone quality indices, including geometric, densitometric, or mechanical properties were observed. Muscle analysis revealed no significant changes in overall meat quality, except for a decrease in cholesterol content and alterations in the fatty acid profile, notably a reduction in total n-3 polyunsaturated and total polyunsaturated fatty acids (PUFA). In conclusion, although not all effects of HMB supplementation were unequivocally beneficial, the positive changes in performance data and trabecular bone microarchitecture support further research into various doses and durations of supplementation. Such studies are necessary to fully understand and optimize the benefits of HMB for enhancing the health and productivity of laying hens.

Raising laying hens: housing complexity and genetic strain affect startle reflex amplitude and behavioural response to fear-inducing stimuli.

Individual variation in fearfulness can be modified during ontogeny, and high levels of fear can affect animal welfare. We asked whether early-life environmental complexity and genetic strain affect fear behaviour in young laying hens (pullets). Four replicates of brown (B) and white (W) genetic strains (breeds) of layers were each raised in four environmental treatments (housing): conventional cages (Conv) and different rearing aviaries with increasing space and complexity (Low < Mid < High). We used a startle reflex test (weeks 4 and 14) to measure startle amplitude and autonomic response (i.e. comb temperature). A combination of novel arena (NA) and novel object (NO) tests was used (week 14) to assess NA exploration and alertness, latency to approach the centre and initial NO avoidance and investigation. Housing × strain affected startle amplitude (B-Conv, B-High < B-Low, B-Mid; B > W; no housing effect in W) but not autonomic response. Fear behaviour was affected by housing (NA exploration, investigation: Conv < Low, Mid, High; NO avoidance: Conv, High < Low, Mid), strain (NA alertness: B > W, NO avoidance: W > B) and their interaction (NA centre approach: B-Conv < all other groups). We present evidence for strain-specific fear responses depending on early experience.

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.

Responses of intestinal morphology, immunity, antioxidant status and cecal microbiota to the mixture of glycerol monolaurate and cinnamaldehyde in laying hens.

This study was to determine the effects of the mixture of glycerol monolaurate and cinnamaldehyde (GCM) supplementation on the intestinal morphology, immunity, antioxidant status and cecal microbiota of laying hens. A total of 1,120 healthy laying hens (Jingfen-1 strain) at the age of 14 wk were randomly divided into 4 groups with 10 replicates of 28 layers in each and layers were fed diets containing 0 (control group), or 250, 500, and 1,000 mg/kg GCM for 12 wk. The results showed that dietary supplementation with GCM significantly increased intestinal villus height and villus height/crypt depth, duodenal villus area, total superoxide disumutase activities in the liver and jejunum, jejunal glutathione peroxidase activities while decreased duodenal and jejunal crypt depth, hydrogen peroxide content in the liver and jejunal malondialdehyde content of laying hens aging 28 wk (P < 0.05). Meanwhile, GCM addition significantly increased serum immunoglobulin A and immunoglobulin M concentration of layers at the age of 20, 24, and 28 wk (P < 0.05). Moreover, it was observed in the 16S rRNA sequencing that the addition of GCM elevated the abundance and diversity of gut microbiota in laying hens. The predominant bacteria from each group were Bacteroidota and Firmicutes at the phylum level and Bacteroides and Lactobacillus were the dominant genera. The composition and structure of cecal microflora were changed by the addition of GCM to the diet of laying hens. In conclusion, the addition of GCM (500-1,000 mg/kg diet) can improve intestinal morphology, immune function, intestinal and liver antioxidant status and intestinal flora of laying hens, thereby improving intestinal digestion and absorption capacity. These findings provide a new way to further explore the mechanism of GCM improving intestinal health.

Prevalence and transmission of extensively drug-resistant Salmonella enterica serovar Kentucky ST198 based on whole-genome sequence in an intensive laying hen farm in Jiangsu, China.

Salmonella, which is widely distributed in nature, is an important zoonotic pathogen affecting humans, livestock, and other animals. Salmonella infection not only hinders the development of livestock and poultry-related industries but also poses a great threat to human health. In this study, we collected 1,537 samples including weak chicks, dead embryos, fecal samples and environmental samples from 2020 to 2023 (for a period of 1 to 2 months per year) to keep a long-term monitor the prevalence of Salmonella in an intensive laying hen farm, 105 Salmonella strains were isolated with an isolation rate of 6.83% (105/1,537). It revealed a significant decrease in prevalence rates of Salmonella over time (P < 0.001). Before 2020, the predominant serotype was S. Enteritidis. S. Kentucky was first detected in November 2020 and its proportion was gradually found to exceed that of S. Enteritidis since then. S. Kentucky isolates were distributed in various links of the four regions in the poultry farm. A total of 55 S. Kentucky strains, were assigned to ST198 based on whole genome sequencing. Among them, 54 strains were resistant to 12 to 16 antibiotics, indicating that they were extensively drug-resistant (XDR). Seventeen antimicrobial resistance genes were detected in 55 S. Kentucky isolates. For most of these isolates, antibiotic resistance phenotypes were concordant with their genotypes. All S. Kentucky strains isolated from this farm in 2020 to 2023 showed a high similarity based on their core-genome SNP-based phylogeny. The traceability analysis revealed that S. Kentucky was introduced to the farm through newly purchased flocks. The long-term existence of XDR S. Kentucky ST198 poses a substantial risk because of the multiage management and circulation of workers in this poultry farm. Thus, this study is the first to report extensively drug-resistant S. Kentucky ST198 detected in this intensive poultry farm in China.

The Impact of Early-Life Cecal Microbiota Transplantation on Social Stress and Injurious Behaviors in Egg-Laying Chickens.

Injurious behaviors (i.e., aggressive pecking, feather pecking, and cannibalism) in laying hens are a critical issue facing the egg industry due to increased social stress and related health and welfare issues as well as economic losses. In humans, stress-induced dysbiosis increases gut permeability, releasing various neuroactive factors, causing neuroinflammation and related neuropsychiatric disorders via the microbiota-gut-brain axis, and consequently increasing the frequency and intensity of aggression and violent behaviors. Restoration of the imbalanced gut microbial composition has become a novel treatment strategy for mental illnesses, such as depression, anxiety, bipolar disorder, schizophrenia, impulsivity, and compulsivity. A similar function of modulating gut microbial composition following stress challenge may be present in egg-laying chickens. The avian cecum, as a multi-purpose organ, has the greatest bacterial biodiversity (bacterial diversity, richness, and species composition) along the gastrointestinal tract, with vitally important functions in maintaining physiological and behavioral homeostasis, especially during the periods of stress. To identify the effects of the gut microbiome on injurious behaviors in egg-laying chickens, we have designed and tested the effects of transferring cecal contents from two divergently selected inbred chicken lines on social stress and stress-related injurious behaviors in recipient chicks of a commercial layer strain. This article reports the outcomes from a multi-year study on the modification of gut microbiota composition to reduce injurious behaviors in egg-laying chickens. An important discovery of this corpus of experiments is that injurious behaviors in chickens can be reduced or inhibited through modifying the gut microbiota composition and brain serotonergic activities via the gut-brain axis, without donor-recipient genetic effects.

Effects of replacing Na selenite in laying hen feed with selenized glucose on production performance, egg quality, egg selenium content, microbial population, immunological response, antioxidant enzymes, and fatty acid composition.

This study aimed to explore the effects of selenized glucose (SeGlu) and Na selenite supplementation on various aspects of laying hens such as production performance, egg quality, egg Se concentration, microbial population, antioxidant enzymes activity, immunological response, and yolk fatty acid profile. Using a 2 × 2 factorial design, 168 laying hens at 27-wk of age were randomly divided into 4 treatment groups with 7 replications. Se source (Na selenite and SeGlu) and Se level (0.3 and 0.6 mg/kg) were used as treatments. When 0.3 mg SeGlu/kg was compared to 0.3 mg Na selenite/kg, the interaction findings revealed that 0.3 mg SeGlu/kg increased egg production percent and shell ash (P < 0.05). When compared to 0.3 mg Na selenite/kg, dietary supplementation with 0.3 and 0.6 mg SeGlu/kg resulted in an increase in albumen height, Haugh unit, and yolk color of fresh eggs (P < 0.05). SeGlu enhanced albumen height, Haugh unit, shell thickness (P < 0.01), albumen index, yolk share, specific gravity, shell ash (P < 0.05) of fresh eggs and shell thickness (P < 0.05) of stored eggs as compared to Na selenite. The interaction showed that 0.6 mg SeGlu/kg enhanced yolk Se concentration while decreasing malondialdehyde levels in fresh egg yolk (P < 0.05). SeGlu enhanced Se concentration in albumen and glutathione peroxidase activity in plasma (P < 0.05) as compared to Na selenite. 0.6 mg Se/kg increased lactic acid bacteria, antibody response to sheep red blood cells, and lowered ∑n-6 PUFA/ ∑n-3 PUFA ratio (P < 0.05). As a result, adding SeGlu to the feed of laying hens enhanced egg production, egg quality, egg Se concentration, fresh yolk lipid oxidation, and glutathione peroxidase enzyme activity.

Effects of dietary supplementation of glycerol monolaurate on laying performance, egg quality, antioxidant capacity, intestinal morphology and immune function in late-phase laying hens.

The objective of this study was to evaluate the effects of different levels of glycerol monolaurate (GML) on laying performance, egg quality, antioxidant capacity, intestinal morphology and immune function in late-phase laying hens. A total of 480 Hy-Line Variety Brown hens (age 54 wk) were randomly assigned to 5 treatments: the control group (basal diet) and 4 GML groups (basal diet supplemented with 100, 200, 300, and 400 mg/kg GML). Each treatment consisted of 8 replicates with 12 hens each and the trial lasted for 8 wk. The results showed that dietary inclusion of GML increased the ADFI in the entire experimental period and the average egg weight in wk 5 to 8 and wk 1 to 8 of the experiment (linear, P < 0.05). Dietary GML addition linearly increased albumen height, Haugh unit and yolk color, and quadratically increased eggshell thickness (P < 0.05). The serum SOD activity, T-AOC and IgG concentrations in the 200 mg/kg GML group, and GSH-Px activity in 200 and 300 mg/kg GML groups were increased, while the MDA concentration in 200 and 300 mg/kg GML groups was decreased than those in the control group (P < 0.05). The jejunal villus height and villus height: crypt depth in 300 mg/kg GML group were higher than that in the control group (P < 0.05). The mRNA expression of TLR4, IL-1ß and TNF-α in spleen and jejunum decreased with the increase of dietary GML concentration (linear, P < 0.05). In conclusion, dietary GML supplementation could improve egg quality, antioxidant capacity, intestinal morphology and immune function in late-phase laying hens, and dietary 300 mg/kg GML inclusion is suggested.

Dietary supplementation of osthole and icariin improves the production performance of laying hens by promoting follicular development.

Osthole (Ost) and icariin (Ica) are extracted from traditional Chinese medicine Cnidium monnieri and Epimedii Folium, respectively, and both exhibit estrogen-like biological activity. This study aimed to determine the efficacy and safety of combining Ost with Ica on the production performance of laying hens and to explore their possible mechanisms. The production performance, egg quality, residues of Ost and Ica in eggs, serum reproductive hormone levels, expression of ovarian reproductive hormone receptor, proliferation of granulosa cells in small yellow follicles (SYF), and progesterone secretion in large yellow follicles (LYF) related genes and proteins expression were detected. The results showed that adding 2 mg/kg Ost + 2 mg/kg Ica to the feed increased the laying rate, average egg weight, Haugh unit, and protein height of laying hens. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4) levels increased, and the expression of ovarian estrogen receptor (ER), follicle-stimulating hormone receptor (FSHR), and progesterone receptor (PGR) mRNA was up-regulated. Additionally, the mRNA and protein levels of steroidogenesis acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) increased in LYF. Furthermore, mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclin E1, and cyclin A2 were up-regulated in SYF. The residues of Ost and Ica in egg samples were not detected by high-performance liquid chromatography (HPLC). In conclusion, dietary supplementation of Ost and Ica increased granulosa cells proliferation in SYF and increased P4 secretion in granulosa cells of LYF, ultimately improving the production performance of laying hens.

ß-Catenin regulates ovarian granulosa cell cycle and proliferation in laying hens by interacting with TCF4.

Ovarian follicle development depends on the proliferation and differentiation of granulosa cells and is a complex biological process. The Wnt/ß-catenin signaling pathway can regulate ovarian follicle development, and ß-catenin, encoded by catenin beta 1 (CTNNB1), is the core component of this pathway. Although several studies of the mechanisms by which the Wnt/ß-catenin pathway regulates cell proliferation in humans and mammals have reported, it remains unclear how ß-catenin functions in poultry. To investigate the function of ß-catenin in laying hens' follicle development, we evaluated the effect of CTNNB1 on cell cycle, proliferation, and apoptosis in ovarian granulosa cells (GCs) isolated from laying hens. We demonstrated that CTNNB1 significantly affected the expression of cyclin D1 (CCND1) and v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) (P < 0.01 and P < 0.05), key genes related to cell cycle and proliferation, to promote cell cycle progression from G1 to S phase, and thus accelerate granulosa cell proliferation. CTNNB1 did not however affect apoptosis or the expression of related genes baculoviral IAP repeat containing 5 (BIRC5) and BCL2 apoptosis regulator (Bcl-2). Overexpression of transcription factor 7-like 2 (TCF4) resulted in increased expression of CCND1, accelerated cell cycle progression, and granulosa cell proliferation. Direct physical interaction between ß-catenin and TCF4 was demonstrated by immunofluorescence and coimmunoprecipitation. The proliferation of granulosa cells was inhibited by silencing CCND1; overexpression of TCF4 in CCND1-silenced cells restored their proliferation rate to normal levels. These results indicate that the interaction of TCF4 and ß-catenin promotes CCND1 expression which in turn accelerates the cell cycle process of laying hen hierarchical follicular granulosa cells.

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.

Influence of keel impacts and laying hen behavior on keel bone damage.

Keel bone damage, which presents as fractures and/or deviations of the keel, has been detected in laying hens housed in all types of systems. Factors leading to keel bone damage in hens housed with limited vertical space, such as those housed in furnished systems, are not well understood, and are the topic of this study. Ten focal hens from each of 12 furnished cages (4 rooms of 3 cages) were fitted with keel mounted tri-axial accelerometers. Their behavior was video recorded continuously over two 3-wk trials: the first when the hens were between 52 and 60 wk of age, and the second approximately 20 wk later. The integrity of each hen's keel was evaluated at the start and end of each 3-wk trial using digital computed tomography. We identified predominant behaviors associated with acceleration events sustained at the keel (collisions, aggressive interactions and grooming) by pairing accelerometer outputs with video data. For each recorded acceleration event we calculated the acceleration magnitudes as the maximum summed acceleration recorded during the event, and by calculating the area under the acceleration curve. A principle components analysis, which was used as a data reduction technique, resulted in the identification of 4 components that were used in a subsequent regression analysis. A key finding is that the number of collisions a hen has with structures in her environment, and the number of aggressive interactions that a hen is involved, each affect the likelihood that she will develop 1 or more fractures within a 3-wk time span. This relationship between hen behavior and keel fracture formation was independent of the magnitude of acceleration involved in the event. Observed behavior did not have an impact on the formation of keel bone deviations, further supporting reports that the mechanisms underlying the 2 types of keel bone damage are different.

Dietary probiotic Lacticaseibacillus paracasei NSMJ56 modulates gut immunity and microbiota in laying hens.

This study was performed to investigate supplementary effects of probiotic Lacticaseibacillus paracasei NSMJ56 strain on laying performance, egg quality, intestinal histology, antioxidant status, gut immunity and microbiota in laying hens. A total of ninety-six 21-wk-old Hy-Line Brown laying hens were randomly subjected to one of 2 dietary treatments: a control group fed a non-supplemented diet, or a probiotic group fed with a diet supplemented with 1 g of Lacticaseibacillus paracasei NSMJ56 (5 × 108 CFU/kg of diet). The trial lasted for 4 wk. Egg weight was increased (P < 0.05) in laying hens fed probiotic-fed diet compared with the control group. Dietary probiotics did not affect egg quality except for Haugh unit, which was improved (P < 0.05) in the probiotic-fed group. Neither jejunal histology nor cecal short-chain fatty acids were affected by dietary treatments. Dietary probiotics increased the activity of catalase compared with the control group. Flow cytometry analysis revealed that dietary probiotics elevated the CD4+ T cells, but not CD8+ T cells, in jejunal lamina propria. Based on the LEfSe analysis at the phylum and genus levels, Erysipelotrichales, Erysipelotrichia, Flintibater, Dielma, Hespellia, Coprobacter, Roseburia, Anaerotignum, and Coprococcus were enriched in the probiotic group compared with the control group. Taken together, our study showed that dietary probiotics could be used to improve some parameters associated with egg freshness and antioxidant capacity, and to partially alter T cell population and microbial community in laying hens.

Integrated transcriptomic and metabolomic analyses reveal potential regulatory pathways regulating bone metabolism pre- and postsexual maturity in hens.

At the onset of sexual maturity, the increasing circulating estrogen stimulates the formation of medullary bone, which provides available calcium for eggshell formation. The bone loss of laying hens is caused by the continuous dynamic changes of structure bone leading to bone fragility and susceptibility. The degree of medullary bone mineralization in sexual maturity is positively correlated with bone quality in the late laying stage. This study aimed to explore the molecular regulation mechanism of bone metabolism pre- and postsexual maturity in hens based on the joint analysis of transcriptome and metabolome. A total of 50 Hy-line Sonia pullets with comparable body weight at 13 wk were selected. Eight pullets were killed at 15 wk (juvenile hens, JH) and 19 wk (laying hens, LH), and LHs were killed within 3 h after oviposition. Differentially expressed genes and metabolites in tibia were analyzed based on transcriptome and metabolome, and then combined to construct the relevant metabolisms and hub genes. In the LH hens, plasma levels of estrogen and tartrate-resistant acid phosphatase were significantly elevated by 1.7 and 1.3 times. In addition, the midpoint diameter, bone mineral density and bone mineral content of the tibia and femur were higher at 19 wk of age. A total of 580 differentially expressed genes were found between the JH and LH group in the tibia, including 280 up-regulated, and 300 down-regulated genes in the LH group. Gene set enrichment analysis (GSEA) showed that the intracellular biosynthesis and secretion of matrix vesicles were significantly enrichment in the LH hens. A total of 21 differential metabolites were identified between JH and LH group. Estradiol valerate positively correlated with L-theanine, tryptophan betaine, dopamine, and perindopril. Joint analysis showed that the top 20 hub genes were enriched in cholesterol biosynthesis and phospholipid metabolism, which played a key regulatory role in bone metabolism during pre- and postsexual maturity. These results provide a theoretical foundation for maintaining efficient egg production and reducing bone health problems in laying hens.