Red junglefowl (RJF) is considered the ancestor of domestic chickens. However, the possible maternal origin, genetic diversity, and subspecies classification of the Philippine (PH) RJF remains uncertain. In this study, the complete mitochondrial DNA (mtDNA) D-loop sequence of 55 PH RJFs collected from the mountainous areas of Occidental Mindoro, Palawan, Agusan del Norte, Capiz, Leyte, Iloilo, and Guimaras were analyzed and compared with chicken reference sequences. Phylogenetic analysis revealed multiple maternal origins of the PH RJFs based on haplogroups D, E, and Y classification. This was supported by PH RJFs and RJFs from other Asian countries sharing a clade. A median-joining network also revealed the haplotype sharing of the PH RJFs and Indonesian RJF, demonstrating common maternal ancestry. High haplotype and nucleotide diversity were also observed at all sampling sites. Analysis of molecular variance indicated that the principal molecular variance existed within populations (81.23%) rather than among populations (18.77%). A population neutrality test and Bayesian skyline plot (BSP) analysis elucidated the RJF maternal effective population size expansion in the Philippines that possibly started approximately 2,800-3,000 years ago. The co-existence of Gallus gallus bankiva and Gallus gallus gallus in the Philippines was also verified. The haplotype sharing of the current RJF samples with commercial chickens suggested the need to formulate conservation programs that would protect the RJFs in the Philippines.

Japanese native chickens (JNCs) comprise approximately 50 breeds, making Japan a diversity hotspot for native chicken breeds. JNCs were established through the repeated introduction of chickens from foreign countries. Jidori, which is the generic name of JNC breeds whose ancestral morphology resembles that of their wild progenitor (red junglefowls), is generally thought to have propagated from north East Asia (Korea and north China) to ancient Japan. However, mitochondrial haplogroup D, which is abundant in Island Southeast Asia (ISEA) as well as the Pacific but relatively rare in other regions, can be observed in some Jidori breeds (e.g., Tosa-Jidori, Tokuji-Jidori) with high frequency, leading to speculation that chickens from ISEA or the Pacific also contributed genetically to JNCs. To test this hypothesis, we sequenced the mitochondrial genomes of Jidori breeds and conducted phylogeographic analysis. Our results indicate that the JNC Haplogroup D belongs to Sub-haplogroup D2, which is currently only observed in Xinjiang, northwest China, and not to Sub-haplogroup D1, which is widely distributed in the ISEA-Pacific region. The other mitochondrial haplogroups of Jidori examined in this study also showed affinity to those of chickens native to north East Asia. Therefore, our findings support the north East Asian origin hypothesis for Jidori.

Body measurement traits (BMTs), which are classical quantitative traits of vital responses to body growth, have been studied in pigs, cattle, and sheep for several decades. In chickens, BMTs mainly cover body slope length, keel length, chest width, chest depth, tibia length, and tibia diameter; however, their genetic markers are yet to be considered. In this study, the Wenshang Barred chicken, a meat-egg-type native breed in China, was used to investigate the association between BMTs and the expression of growth-related genes, including GH, IGF1, IGF2, GHRL, IGF1R, IGFBP2, GHF-1, and TSHB. The results revealed that the single nucleotide polymorphism (SNP) rs3138025 in GH was significantly associated with keel length (P＝0.0455 <0.05), rs313810945 in IGF2 was significantly correlated with chest width (P＝0.0454 <0.05) and chest depth (P＝0.0259 <0.05), and rs317298536 in TSHB significantly affected chest depth (P＝0.0399 <0.05). The SNPs were associated with traits reflecting body size and were potentially involved in bone growth, which was consistent with studies in humans, rodents, and other vertebrate species. In addition, a borderline significant association was found between rs317298536 and body weight (P＝0.0604). These polymorphic sites may be treated as candidate genetic markers in breeding programs involving Wenshang Barred chickens.

The pullet phase is an important stage in the development of laying hens when the development of organs, including reproductive organs and bones, is rapid. However, in recent years, few studies have focused on this crucial stage. The purpose of this study was to evaluate the effect of a dietary crude protein (CP) regimen during the rearing period (9-21 weeks (wks) of age) on pullet development and the subsequent performance, egg quality, and bone quality of Hy-Line Brown laying hens. A total of 256 pullets were randomly assigned to two treatments. Each treatment was replicated eight times with 16 pullets per replicate (n＝8), which were fed ad libitum using either of the two CP regimens: (1) 14%-18% CP (fed with 14% and 18% CP from 9-17 wks and 18-21 wks, respectively); (2) 16% CP (fed with 16% CP from 9-21 wks of age). At 21 wks of age, eight birds per treatment were randomly selected to evaluate body composition and ovarian development. For quality analysis, eggs were collected at 28, 32, 36, and 70 wks. At 70 wks of age, eight hens per treatment were selected to evaluate bone quality. There were no treatment differences in pullet performance, body composition, and ovarian development at 21 wks. The dietary CP regimen during the rearing period (9-21 wks) did not influence laying performance during the laying period. There were no treatment differences in tibial and femoral quality at 70 wks. Egg quality results showed an inconsistent trend. It was concluded that the pullets fed with the low CP grower diet (14%) during the pullet period and a high CP pre-lay diet (18%) from 18-21 wks of age developed properly and had satisfactory laying performance. However, the rearing diet did not enhance bone quality.

Smooth muscle cells are widely distributed in the digestive organs of chickens. They exist as single cells, but adhere to each other to function synchronously. In this study, the expression of the gap junction protein connexin 43 (Cx43) in chicken gizzards was investigated at embryonic days (E) 10, E15, and E18. Gizzards have an abundance of smooth muscle cells because of their thick muscle layers, which enable easy analysis of the cells. Morphological observations and expression patterns of smooth muscle markers were confirmed. Next, we observed where the markers were localized in the gizzard tissue at E10, E15, and E18. Finally, the expression pattern of Cx43 in primary cultured smooth muscle cells from E15 gizzards was investigated. The analysis revealed the expression and localization of Cx43 and calponin 1 in the smooth muscle layers, and 3D analysis revealed dynamic changes in the localization pattern of Cx43 from E10 to E15. Cultured smooth muscle cells confirmed that Cx43 was expressed in the cell membrane and cytosol. In conclusion, Cx43 expression was identified in chicken gizzards at E10, E15, and E18, which was localized differently during development. The expression was broad at E10, and became restricted at E15 and E18. Primary culture of smooth muscle cells showed that Cx43 was present in the cell membrane and cytosol. This suggests that Cx43 is actively translated into the cytosol at E10, forming a hexamer, and shuttling the cell membrane to function as a gap junction.

Elucidating taste sensing systems in chickens is an important step toward understanding poultry nutrition. Amino acid taste receptors, type 1 taste receptors 1 and 3 (T1R1 and T1R3, respectively), are expressed in chicken taste cells, and chicken T1R1/T1R3 is activated by L-alanine (L-Ala) and L-serine (L-Ser), but not by L-proline (L-Pro). However, it is not clear whether chickens have a gustatory perception of L-amino acids. Here, we found that chickens conditioned to avoid either L-Ala, L-Ser, or L-Pro solutions could successfully learn to avoid the corresponding L-amino acid solution in the conditioned taste aversion (CTA) test. Because CTA is a well-established learning paradigm generated specifically by pairing gustatory perception and gastrointestinal malaise, the present study suggests that chickens can sense L-amino acids by gustatory perception. In addition, we found that the expression of the T1R1 and T1R3 genes was significantly downregulated in response to chronic exposure to L-Ala solution, but not to acute oral stimulation. Taken together, the present study suggests that chickens have a gustatory perception of L-amino acids, and the expression of T1R1/T1R3 mRNAs in the oral cavity can be regulated by L-amino acid intake. Since chickens can detect L-Pro solutions, additional amino acid receptors, other than T1R1/T1R3, may be involved in L-amino acid taste detection in chickens.

Previous studies in mammalian obesity models have suggested that central transforming growth factor-β (TGF-β) controls the gene expression of appetite-regulating neuropeptides and peripheral energy metabolism. In the present study, we investigated the possible involvement of central TGF-β/Smad signaling in feeding regulation in chickens. Central administration of TGF-β1 resulted in phosphorylation of Smad2 in the hypothalamus of chicks and suppressed feed intake without changing the gene expression of hypothalamic appetite-regulating neuropeptides (neuropeptide Y, agouti-related protein, proopiomelanocortin, and corticotropin-releasing factor). However, neither fasting nor refeeding induced the phosphorylation of hypothalamic Smad2. These findings suggest that the activation of hypothalamic TGF-β/Smad signaling suppresses feed intake in chicks but it might not occur in response to feeding status.

Difference of onset of increase of PRL content in the anterior pituitary gland and plasma PRL concentration during the late stage of chicken embryogenesis is well known. To investigate the disagreement, changes in PRL content and PRL mRNA levels, and the effects of vasoactive intestinal polypeptides (VIP) on PRL release and PRL mRNA expression were examined using western blot analysis and real-time PCR quantification. Changes in SPRL content were strongly correlated with PRL mRNA levels. The increase in PRL content on day 17 of incubation may be caused by the increase in PRL mRNA levels on day 16 of incubation. Additionally, the effects of VIP on PRL release from the embryonic anterior pituitary gland were not observed until day 18 of embryogenesis. These results suggest that increased levels of PRL mRNA and PRL content in the anterior pituitary gland are closely correlated. However, the increased expression of PRL mRNA observed on day 17 and the initiation of PRL release from the anterior pituitary gland on day 19 were differentially regulated. According to the results of western blot analysis, the proportion of glycosylated PRL (G-PRL) and non-glycosylated PRL (NG-PRL) in the anterior pituitary gland at the end stage of development differed from the proportion of PRL released from the anterior pituitary gland. According to the results of two-dimensional western blot analysis, no isoforms with different isoelectric points were detected in the culture medium on days 19 and 20. These data suggest that the peptide chains of G-PRL and NG-PRL were not modified. In conclusion, the differentiation of PRL-producing cells and the maturation of the hypothalamus and anterior pituitary gland were completed at the end stage of incubation, and that different factors regulated the initiation of PRL mRNA expression before day 18 of incubation.

The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P<0.05), while the plasma calcium concentrations were significantly lower (P<0.05) than those in the intact control (IC) group. However, no significant differences in embryo viability and other major blood component levels were observed among the experimental groups (P>0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P<0.05) embryos. Severe renal tubular degeneration of the metanephros was observed in D19-cSLC embryos despite oxygen gas injection starting from D17. These results indicate that D19-cSLC embryos are hypoxia even after injecting oxygen gas starting on D17.

This study investigated whether freezing or warming water-in-oil (W/O) vaccines affected the immune responses of chickens. One of the conditions affecting the efficacy of commercially available animal vaccines is the storage temperature range. Previous studies have shown that the properties of some inactivated vaccines change owing to freezing, leading to reduced immune responsiveness after inoculation. In this study, we first determined the freezing temperatures of a commercial W/O vaccine using freezers maintained at −10, −13, −15, and −20°C. The results showed that the W/O vaccine froze from −10 to −12°C. Next, we evaluated the effect on antibody level transitions (sample-to-positive ratio) in 46-day-old broiler chickens vaccinated with the W/O vaccine that was maintained at −20°C, 5°C, and −10°C, in that order. In addition, the effect on antibody value transitions was evaluated in 45-day-old broiler chickens vaccinated with the W/O vaccines that were frozen and thawed between −20°C and 5°C repeatedly or warmed to 42°C. In these experiments, no remarkable effect of the freeze-thawing or warming treatments on antibody value transitions was observed. These results suggested that the efficacy of the W/O vaccine was not significantly affected when placed in a frozen environment or left in a room temperature environment of 42°C or lower for approximately 5 d. These data indicate the possibility of expanding the temperature range for handling W/O vaccines.

Amyloid fibrils, which are formed from aggregates of aberrant proteins, can cause various forms of amyloidosis (including Alzheimer’s disease). Such disorders often occur in elderly populations and are suspected to be lifestyle related. Thus, it has been speculated that some foodstuffs could be beneficial for preventing amyloidosis. In this study, we determine whether fibril formation by the hen egg white lysozyme (HEWL) could be inhibited by conducting a thioflavin T assay followed by fluorescence and electron microscopy observations. The results demonstrated that four peptide specimens prepared by the hydrolysis of crude proteins from the egg white, egg yolk, chalazae, and eggshell membrane of hen eggs effectively inhibited HEWL fibril formation. Among the four specimens, peptides from chalazae exhibited the highest preventive ability. The superiority of chalaza peptides was also observed when fibril formation was assayed using a full-length human lysozyme and human amyloid β peptide 1-42, which is the key factor for the development of Alzheimer’s disease. Our study of the fibrillization of the human lysozyme also showed that metal ions (Zn²⁺, Ca²⁺, Co²⁺, Mn²⁺ and Al³⁺) promoted fibrillization, and their effects were abolished by the peptide specimens (especially by chalaza peptides). Thus, we conclude that chicken-egg proteins could be a convenient source of therapeutic materials for amyloidosis.