In vitro fertilization has been widely used to produce offspring in several mammalian species. We previously successfully produced Japanese quail chicks using intracytoplasmic sperm injection (ICSI), whereas in vitro insemination was not successful. This may be due to the difficulties associated with mimicking the sperm-egg fusion process and subsequent events in physiological polyspermic fertilization in vitro. In the present study, we observed egg development after in vitro insemination and investigated the inactivation of metaphase-promoting factor (MPF) and cytostatic factor (CSF), which are downstream of the Ca²⁺ signaling pathway in the egg, due to fertilizing sperm. We found a sperm number-dependent increase in hole formation caused by sperm penetration of the perivitelline membrane, the extracellular coat surrounding the egg. Egg development was observed following in vitro insemination; however, the developmental rate and stages after 24-h culture were inferior to those of ICSI eggs, even when insemination was performed with a high number of sperm (2 × 10⁴). We also noted the downregulation of inositol 1,4,5-trisphosphate receptor-1, ryanodine receptor-3, cyclin B1, and c-MOS, which are important regulatory components of MPF and CSF in the egg, which was dependent on the number of sperm used for insemination. However, the decreases observed in these components did not reach the levels observed in the ICSI eggs. Collectively, the present results suggest that a sperm number higher than 2 × 10⁴ is required for the progression of the Ca²⁺ signaling pathway, which initiates subsequent egg development in Japanese quail.

CpG-oligodeoxynucleotides (CpG-ODNs ) have been shown to possess immunostimulatory features in both mammals and birds. However, compared to their proinflammatory effects,little is known about the anti-inflammatory responses triggered by CpG-ODN in avian cells. Hence, in this study, the anti-inflammatory response in the chicken macrophage cell line HD11 was characterized under stimulation with five types of CpG-ODNs: CpG-A₁₅₈₅, CpG-A_D35, CpG-B₁₅₅₅,CpG-B_K3, and CpG-C₂₃₉₅. Single-stimulus of CpG-B₁₅₅₅, CpG-B_K3, or CpG-C₂₃₉₅ induced interleukin (IL)-10 expression without causing cell injury. The effects of pretreatment with CpG-ODNs before subsequent lipopolysaccharide stimulation were also evaluated. Interestingly, pretreatment with only CpG-C₂₃₉₅ resulted in high expression levels of IL-10 mRNA in the presence of lipopolysaccharide. Finally, gene expression analysis of inflammation-related cytokines and receptors revealed that pre-treatment with CpG-C₂₃₉₅ significantly reduced the mRNA expression of tumor necrosis factor-α,IL-1β, IL-6, and Toll-like receptor 4.Overall, these results shed light on the anti-inflammatory responses triggered by CpG-C₂₃₉₅ stimulation through a comparative analysis of five types of CpG-ODNs in chicken macrophages. These results also offer insights into the use of CpG-ODNs to suppress the expression of proinflammatory cytokines, which may be valuable in the prevention of avian infectious diseases in the poultry industry.

Silicate minerals are common additives in poultry feed. To assess their effects, we added zeolite (ZEO) and methyl-sulfonyl-methane (MSM) to broiler chicken diets. A total of 960 one-day-old Ross broiler chicks were randomly divided into four dietary groups with six replicates. Each broiler was maintained until it reached 35 days of age. A completely randomized 2 × 2 experimental design was used, with two ZEO (0 and 1.0%) and two MSM (0 and 0.10%) levels. We observed an additive effect (P<0.05) on interleukin-2 (IL-2) concentrations in broiler bursa and serum when both ZEO and MSM were present. Both ZEO or MSM produced significant (P<0.05) increases in body weight, weight gain, and feed intake. Both increased IL-2 and IL-6 levels in the bursa and serum. Neither affected the serum concentrations of albumin, AST, cholesterol, HDL cholesterol, glucose, total protein, or triglycerides. In summary, these results support supplementation with ZEO and MSM in broiler diets, both separately and in combination.

Ornithine has been identified as a potential satiety signal in the brains of neonatal chicks. We hypothesized that brain nutrient signals such as amino acids and appetite-related neuropeptides synergistically regulate food intake. To test this hypothesis, we investigated the interaction between neuropeptide Y (NPY) and ornithine in the control of feeding behavior in chicks and the associated central and peripheral amino acid metabolic processes. Five-day-old chicks were intracerebroventricularly injected with saline, NPY (375 pmol), or NPY plus ornithine (2 or 4 μmol) at 10 μl per chick, and then subjected to ad libitum feeding conditions; food intake was monitored for 30 min after injection. Brain and plasma samples were collected after the experiment to determine free amino acid concentrations. Co-injection of NPY and ornithine significantly attenuated the orexigenic effect induced by NPY in a dose-dependent manner. Central NPY significantly decreased amino adipic acid, asparagine, γ-aminobutyric acid, leucine, phenylalanine, tyrosine, and isoleucine levels, but significantly increased lysine levels in the brain. Co-injection of NPY and ornithine significantly increased ornithine and proline levels in all examined brain regions, but decreased diencephalic tryptophan and glycine levels compared with those of the control and NPY-alone groups. Co-injection of NPY and high-dose ornithine significantly decreased methionine levels in all brain regions. Central NPY significantly suppressed the plasma concentrations of amino acids, including proline, asparagine, methionine, phenylalanine, tyrosine, leucine, isoleucine, glycine, glutamine, alanine, arginine, and valine, and this reduction was greater when NPY was co-injected with ornithine. These results suggest that brain ornithine interacts with NPY to regulate food intake in neonatal chicks. Furthermore, central NPY may induce an anabolic effect that is modified by co-injection with ornithine.

The aim of this study was to determine whether Newcastle disease/infectious bronchitis (ND/IB) vaccination and yeast product diet supplementation modulate the expression of innate immune molecules in the proventriculus and ileum of broiler chicks. One-day-old male broiler chicks were divided into four groups (V–Y– (control), V–Y+, V+Y–, and V+Y+ groups, where V and Y represent vaccination and yeast product supplementation, respectively). Chicks in the V+Y– and V+Y+ groups were immunized with the live ND/IB vaccine, whereas chicks in the V–Y– and V–Y+ groups were not. Chicks in the V–Y+ and V+Y+ groups received feed containing yeast products from day 4, whereas chicks in the V–Y– and V+Y– groups did not. The proventriculus and ileum were collected on day 7 to analyze the expression of seven Toll-like receptors (TLRs) and Dectin-1. In the proventriculus, compared with those of the V–Y– control group, the TLR7 and TLR21 expression levels were higher in the V+Y– group; however, there were no differences in the expression levels of any TLR or Dectin-1 in the ileum. There were also no differences in the expression of avian β-defensins and cathelicidin-1 in the proventriculus and ileum between the control and treatment groups. The expression of granzyme in cytotoxic cells and interleukin (IL)-1B was upregulated by ND/IB vaccination in the proventriculus. Supplementation with yeast products upregulated only granzyme expression in the ileum and downregulated IL-6 expression in the proventriculus in chicks immunized with the ND/IB vaccine. Thus, we concluded that ND/IB vaccination is effective at enhancing the innate immune system in the proventriculus of chicks, at least until day 7 post-hatching, whereas the effects of diet supplementation with yeast products may be limited, at least under the present study conditions.

Vitamin E is an essential micronutrient for animals. The aim of this study was to determine the effect of vitamin E on intramuscular fat (IMF) deposition and the transcriptome profile of the pectoral muscle in broiler chickens. Arbor Acres chickens were divided into five treatment groups fed a basal diet supplemented with 0, 20, 50, 75, and 100 IU/kg dietary DL-α-tocopheryl acetate (vitamin E), respectively. Body weight, carcass performance, and IMF content were recorded. Transcriptome profiles of the pectoral muscles of 35-day-old chickens in the control and treatment groups (100 IU/kg of vitamin E) were obtained by RNA sequencing. The results showed that diets supplemented with 100 IU/kg of vitamin E significantly increased IMF deposition in chickens on day 35. In total, 159 differentially expressed genes (DEGs), including 57 up-regulated and 102 down-regulated genes, were identified in the treatment (100 IU/kg vitamin E) group compared to the control group. These DEGs were significantly enriched in 13 Gene Ontology terms involved in muscle development and lipid metabolism; three signaling pathways, including the mitogen-activated protein kinase and FoxO signaling pathways, which play key roles in muscular and lipid metabolism; 28 biofunctional categories associated with skeletal and muscular system development; 17 lipid metabolism functional categories; and three lipid metabolism and muscle development-related networks. The DEGs, pathways, functional categories, and networks identified in this study provide new insights into the regulatory roles of vitamin E on IMF deposition in broilers. Therefore, diets supplemented with 100 IU/kg of vitamin E will be more beneficial to broiler production.

In recent years, the market share for cage-free eggs has gradually increased. Because commercially available cage-free eggs are often produced not only by several housing systems but also with different feed crude protein (CP) levels, there are combined effects of feed and housing systems between cage-free and cage eggs. Therefore, using field data, this study aimed to determine the combined effects of feed and housing systems on egg traits and yolk and albumen amino acids in table eggs. Brown layers (n = 40) at the middle laying stage under two feed and housing systems (cage, CP 15.5% diet; barn, CP 17.0% diet) were used. One-way analysis of variance and Pearson’s correlation analysis were used to evaluate 10 egg traits, 19 yolk amino acid traits, and 20 albumen amino acid traits. We observed significant effects of feed and housing on two egg traits (yolk weight and eggshell color redness), 16 yolk amino acids (Asp, Glu, Asn, Ser, Gln, His, Arg, Thr, Ala, Tyr, Met, Cys, Ile, Leu, Phe, and Lys), and 14 albumen amino acids (Asp, Asn, Ser, Gln, Gly, His, Arg, Thr, Ala, Val, Met, Cys, Ile, and Leu). This study revealed that eggs from the barn system (CP 17.0%) contained higher levels of free amino acids in 15 yolk and nine albumen amino acid traits. Phenotypic correlations among the 49 egg traits indicated similar correlation patterns in both systems, which implies that the balance of free amino acid content in yolk and albumen is similar in each system. Although some potential confounding factors may be present for comparing egg content between cage (CP 15.5%) and barn (CP 17.0%) systems, this study suggests that commercially available cage-free eggs may be different from cage eggs not only in external egg traits but also yolk and albumen amino acid traits.