The mucosa of the intestine and oviduct of hens are susceptible to pathogens. Pathogenic infections in the mucosal tissues of laying hens lead to worsened health of the host animal, decreased egg production, and bacterial contamination of eggs. Therefore, better understanding of the mechanisms underlying mucosal barrier function is needed to prevent infection by pathogens. In addition, pathogen infection in the mucosal tissue generally causes mucosal inflammation. Recently, it has been shown that inflammation in the oviduct and intestinal tissue caused by disruption of the mucosal barrier function, can affect egg production. Therefore, it is vitla to understand the relationship between mucosal barrier function and egg production to improve poultry egg production. This paper reviews the studies on (1) oviductal mucosal immune function and egg production, (2) intestinal inflammation and egg production, and (3) improvement of mucosal immune function by probiotics. The findings introduced in this review will contribute to the understanding of the mucosal barrier function of the intestine and oviduct and improve poultry egg production in laying hens.

Fertilization requires successful completion of molecular events taking place at different spatiotemporal scales. Transcriptionally and translationally inactive sperm need to rely on pre-assembled pathways modulated by extracellular signals that traverse the plasma membranes. However, species differences in how sperm respond to them delay the progress toward a comprehensive understanding of how activation of the signaling cascades is coordinated in poultry sperm. In chickens, recent studies have found that membrane rafts are present on the sperm surface and play important roles in regulating multistage fertilization. In this review, we focus on three steps in which membrane alteration plays a key role. The first is post-testicular maturation, in which bird sperm acquire fertilization functions through biochemical changes. The second part of this review concerns membrane regulation of sperm-egg binding and the acrosome reaction. Finally, we extend our discussion to the translation of membrane raft theory into a technical principle for the commercial production and genetic preservation of poultry.

Upon contact with laid eggs, avians initiate incubation behavior and stop laying additional eggs. This phenomenon suggests that the productivity of laying hens in free-range facilities may decrease because of frequent contact with laid eggs. Here, we examined whether hens of a commercial breed exhibit incubation behavior in a free-range facility and whether egg productivity subsequently decreases. One-hour observations were performed twice weekly for 3 weeks, during which 9 of 129 hens (7.0%) exhibited incubation behavior (i.e., sitting on eggs) in the free-range facility and were defined as incubating hens. During 4 d of continuous behavioral observation, incubating and non-incubating hens laid the same number of eggs statistically (4.6 and 3.6, on average, respectively); however, incubating hens spent significantly more time on average incubating the eggs (2071.9 min) than did the non-incubating hens (20.9 min; P<0.05), indicating a clear behavioral difference. Subsequently, the incubation behavior and egg productivity of incubating hens and a Silkie Fowl breed hen, which is known to exhibit typical incubation behavior and cessation of laying, were continuously compared for 27 d. The average minutes spent incubating eggs during the observation period increased in both the incubating hens and Silkie Fowl hen and the total time was almost the same (18,088.5 and 23,092 min, respectively). However, the Silkie Fowl hen stopped laying on day 17 after laying 17 eggs, whereas the incubating hens continued laying throughout the observation period. Incubating hens laid an average of 24.5 eggs, indicating that some hens (at least those of the commercial breed used in our study) can continue laying while exhibiting incubation behavior. A single-nucleotide polymorphism associated with incubation behavior was detected on chromosome 4 through genome-wide association analysis.

1,25-Dihydroxycholecalciferol (1,25-(OH)₂-D₃) is the final active product of vitamin D. This study aimed to investigate the effects of 1,25-(OH)₂-D₃ on growth performance, bone development, and calcium (Ca) transporter gene expression levels in the small intestine of broiler chickens. On the day of hatching, 140 female Ross 308 broilers were randomly allotted into two treatments with five replicates (14 birds per replicate). Two levels of 1,25-(OH)₂-D₃ (0 and 1.25 µg/kg) were added to the basal diet without vitamin D. Results showed that the addition of 1.25 µg/kg 1,25-(OH)₂-D₃ increased the average daily feed intake and the average daily gain and decreased the feed conversion ratio and mortality in 1- to 19-day-old broiler chickens compared with the basal diet without vitamin D (P<0.05). 1,25-(OH)₂-D₃ also enhanced the length, weight, ash weight, and the percentage contents of ash, Ca, and P in the tibia and femur of broilers (P<0.05). The mRNA expression levels of the Ca-binding protein (CaBP-D28k) in the duodenum, jejunum, and ileum of 19-day-old broilers increased to 88.1-, 109.1-, and 2.7-fold, respectively, after adding 1,25-(OH)₂-D₃ (P<0.05). The mRNA expression levels of the plasma membrane Ca ATPase 1b (PMCAlb) in the duodenum and the sodium (Na)/Ca exchanger 1 (NCX1) in the duodenum and the jejunum were also enhanced to 1.57-2.86 times with the addition of 1,25-(OH)₂-D₃ (P<0.05). In contrast, the mRNA expression levels of PMCA1b and NCX1 in the ileum and that of vitamin D receptor (VDR) in the small intestine were not affected by 1,25-(OH)₂-D₃ (P>0.05). These data indicate that 1,25-(OH)₂-D₃ upregulated Ca transporter gene transcription and promoted Ca²⁺ absorption in the small intestine, especially in the proximal intestine (duodenum and jejunum), thereby improving growth performance and bone mineralization in broiler chickens.

The effects of dietary carotenoids on egg yolk were investigated in this study. Forty Rhode Island Red (RR) and 40 Silky Fowl (SF) hens that were 60 weeks old were used. Hens of each breed were randomly divided into four dietary groups. One group was fed a basal diet (crude protein 17%, metabolizable energy 2800 kcal/kg) only, whereas the other groups received a specific additive, namely, paprika extract, marigold petal extract, or Paracoccus cell powder, in addition to the same basal diet. The color and carotenoid content of egg yolk and singlet oxygen quenching activity were measured after 4 weeks. The total carotenoid content, zeaxanthin content, and singlet oxygen quenching activity in the yolk differed significantly between breeds and between diets (two-way ANOVA). The lutein content in egg yolk was affected by breed and diet, as well as by the interaction between these two factors. Regarding the Roche Yolk Color Fan values, only the effect of diet was significant. In terms of objective egg yolk color, there was a significant difference in lightness and yellowness between breeds. The total carotenoid content was higher in SF than in RR in all the groups. Likewise, the levels of zeaxanthin and lutein in the yolk were higher in SF than in RR (P<0.05). The results of the present study suggest that dietary carotenoids are effective feed additives for laying hens, especially SF, to improve the color and singlet oxygen quenching activity of egg yolk.

Three experiments were conducted to determine the metabolizable energy (ME) and net energy (NE) values of expanded cottonseed meal (ECSM) for broilers aged 14-16 days (Experiment 1), broilers aged 28-30 days (Experiment 2), and 45-week-old Hy-Line Brown hens (Experiment 3). Reference diets based on corn-soybean meal were used to meet the nutritional needs of the birds. The test diets contained ECSM as basis, which was used to replace 18.5% of the gross energy-yielding ingredients from the reference diet. The birds were fed a commercial feed before the experimental period. After the dietary adaptation period, six birds per replicate (Experiment 1) and two birds per replicate (Experiments 2 and 3) for each treatment group were placed in an individual open-circuit respiratory calorimetry chamber for 3 days. Daily O₂ consumption and CO₂ production were recorded, and excreta samples were collected. The ME and NE values of ECSM were determined using the substitution method. The apparent metabolizable energy (AME) values of ECSM for experiments 1, 2, and 3 were 2605.85, 2178.31, and 2782.60 kcal/kg of dry matter (DM), respectively. The NE values were 1655.23, 1196.64, and 1538.19 kcal/kg of DM, respectively. The NE:AME ratios of ECSM were 63.52%, 54.93%, and 55.29%, respectively. Our data showed that the ME and NE values of ECSM differed across various growth stages and types of chickens. These results demonstrate that the appropriate ME and NE should be used in the design of different feed formulas for specific growth stages and types of chickens.

Excessive accumulation of body fat in broiler chickens has become a serious problem in the poultry industry. However, the molecular mechanism of triglyceride accumulation in chicken white adipose tissue (WAT) has not been elucidated. In the present study, we investigated the physiological importance of the catabolic hormone corticosterone, the major glucocorticoid in chickens, in the regulation of chicken WAT lipid metabolism. We first examined the effects of fasting on the mRNA levels of lipid metabolism-related genes associated with WAT, plasma corticosterone, and non-esterified fatty acid (NEFA). We then examined the effects of corticosterone on the expression of these genes in vivo and in vitro. In 10-day-old chicks, 3 h of fasting significantly decreased mRNA levels of lipoprotein lipase (LPL) in WAT and significantly elevated plasma concentrations of NEFA. Six hours of fasting significantly increased mRNA levels of adipose triglyceride lipase (ATGL) in WAT and significantly elevated plasma concentrations of corticosterone. On the other hand, fasting significantly reduced mRNA levels of LPL in WAT and elevated plasma concentrations of NEFA in 29-day-old chicks without affecting mRNA levels of ATGL in WAT or plasma corticosterone concentrations. Oral administration of corticosterone significantly reduced mRNA levels of LPL and significantly increased the mRNA levels of ATGL in WAT in 29-day-old chicks without affecting plasma NEFA concentrations. The addition of corticosterone to primary chicken adipocytes significantly increased mRNA levels of ATGL, whereas mRNA levels of LPL tended to decrease. NEFA concentrations in the culture medium were not influenced by corticosterone levels. These results suggest that plasma corticosterone partly regulates the gene expression of lipid metabolism-related genes in chicken WAT and this regulation is different from the acute elevation of plasma NEFA due to short-term fasting.

The ostrich (Struthio camelus) is an herbivorous bird with a long and developed hindgut. In the hindgut, there is a dense and highly diverse population of anaerobic bacteria, and active fermentation produces high concentrations of short-chain fatty acids. Bacteria in the hindgut of the ostrich are considered vital for both their nutritional contribution and health benefits, such as benefits to the immune and defense system of the host. We attempted to isolate Lactobacillaceae, which might be involved in improving immune function and in inhibiting pathogens. The number of colonies from ostrich feces observed on LBS agar medium was 3.64×10³ per gram of feces. Three strains of Lactobacillaceae were isolated from the feces. Nearly the entire length of the 16S ribosomal RNA gene of these isolates was sequenced, and a homology search showed high identity with L. brevis (identity＝99.93%), L. coryniformis (98.39%), and L. paracasei (100.0%). These isolates may be deemed potential probiotics for the ostrich.

Skin thickness and strength differ between male and female chickens. This study aimed to clarify the effects of estradiol on the expression of estrogen receptors and collagen mRNA in chicken skin. Estradiol was administered to male chicks for 3 weeks, then cryosections of skin collected from the cervical, thoracic, dorsal, and pelvic limb regions were stained with hematoxylin and eosin, and dermal thickness was measured. Estrogen receptor and collagen mRNA expression was assessed using real-time RT-PCR, and collagen contents were determined. Estradiol did not alter dermal thickness or the collagen content of the skin from any tested region. Among the estrogen receptors, significantly more ESR1 mRNA was expressed in the thoracic skin of chicks administered with estradiol compared with vehicle (control), and in the thoracic skin compared with skin from other regions within each group. Estradiol did not affect ESR2, GPER, and COL1A1 mRNA expression. These results suggested that estradiol stimulates ESR1 expression in thoracic skin, but does not affect collagen synthesis in skin from any other region of male chicks.

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca²⁺ ([Ca²⁺]i): transient elevations in [Ca²⁺]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca²⁺ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca²⁺]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca²⁺ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca²⁺]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca²⁺ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate increases in [Ca²⁺]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca²⁺ wave and spiral-like Ca²⁺ oscillations, respectively.

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 μg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund’s complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16-19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.