The avian immune system plays a vital role in poultry production to obtain good productibility and products that are safe and of high quality. Historically, adaptive immunity has been the main target of vaccination. However, over the past decade, innate immunity has been reported to be enhanced in different animals through vaccination and feed additives. This enhancement is due to innate immune memory termed “trained immunity,” in which epigenetic and metabolic reprogramming play significant roles. Although reports on trained immunity in poultry are limited, several studies have suggested that vaccinations and feed additives affect the innate immunity. This review discusses the possible effects of vaccination and β-glucan on innate immunity for potential incorporation in advanced strategies to enhance the defense function in poultry while considering the information on trained immunity in mammals.

Food intake affects poultry productivity. A complete understanding of these regulatory mechanisms provides new strategies to improve productivity. Food intake is regulated by complex mechanisms involving many factors, including the central nervous system, gastrointestinal tract, hormones, and nutrients. Although several studies have been conducted to elucidate regulatory mechanisms in chickens, the mechanisms remain unclear. To update the current knowledge on feeding regulation in chickens, this review focuses on recent findings that have not been summarized in previous reviews, including spexins, adipokines, neurosecretory proteins GL and GM, and central intracellular signaling factors.

This study aimed to measure the effects of trehalose (Tre) supplementation on the growth, intestinal morphology, gut bacteria, and footpad dermatitis (FPD) of broiler chickens reared at different stocking densities (SD). Four hundred newly hatched Ross 308 male chicks were randomly allocated to four groups of eight, following a 2 × 2 factorial arrangement in a randomized complete block design using two SDs (normal, 11; high, 14 birds/m²) and two diets: basal with and without 0.5% Tre. Tre supplementation was provided during the starter/grower phase, but not the finisher phase. Data were analyzed using a two-way analysis of variance. We observed no significant effects of SD or Tre, individually or combined, on body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) during the starter/grower period. However, high SD decreased both BWG (P < 0.001) and FI (P < 0.05), and increased FCR (P < 0.001), during the finisher period. Whereas Tre reduced FCR (P < 0.05) as a main effect, no combined effect was observed on FCR. Over the total period, high SD negatively affected BWG and FCR (P < 0.001), and Tre significantly reduced FCR, with its effect unaffected by SD. No significant effects of SD or Tre were observed on jejunal morphology. The ileal abundance of Clostridium perfringens (P > 0.05) was not affected by high SD but was significantly reduced by Tre. Neither high SD nor Tre altered Lactobacillus spp. counts; however, high SD increased FPD lesion scores, whereas Tre had no effect. The study showed that Tre supplementation during the starter/grower period improved FCR during the finisher period, possibly by decreasing the abundance of C. perfringens in broiler chickens.

Puerarin is an isoflavone extracted from Gegen (Pueraria lobata) and has been widely utilized to treat various human diseases; however, information regarding its benefits in animal production is limited. In this study, we aimed to investigate the influence of dietary puerarin supplementation on growth performance, immune organ index, immunoglobulin profile, antioxidant capacity, and intestinal morphology in pigeons. In total, 375 healthy 28-day-old White King pigeons were randomly divided into five groups, each consisting of five replicates and 15 pigeons per replicate. Each group was administered one of five dietary treatments: the basal diet, or the basal diet supplemented with 40, 80, 120, or 160 mg/kg puerarin. Treatment duration was 30 days following a 7-day acclimation period. Puerarin treatment did not significantly alter the growth performance of pigeons but afforded a significant linear enhancement in the thymus index (P < 0.05). Additionally, puerarin supplementation significantly increased serum immunoglobulin A and immunoglobulin M levels in pigeons in a linear manner (P < 0.05). Similarly, puerarin significantly and linearly increased the activities of total antioxidant capacity, superoxide dismutase, glutathione, and catalase in the serum and liver, and decreased the malondialdehyde content (P < 0.05). Moreover, the villus height (VH), crypt depth (CD), and VH/CD ratio of the small intestine (including the duodenum, jejunum, and ileum) increased linearly upon puerarin supplementation (P < 0.05). Collectively, these results indicate that puerarin supplementation could improve the immune response, antioxidant capacity, and intestinal morphology of pigeons.

Imidazole dipeptides possess important bioregulatory properties in animals. This study aimed to evaluate the effect of high ambient temperature on muscle imidazole dipeptides (carnosine, anserine, and balenine) in broiler chickens. Sixteen 14-day-old male broiler chickens were divided into two groups, which were reared under thermoneutral (25 ± 1 °C) or cyclic high ambient temperature (35 ± 1 °C for 8 h/day) for 4 weeks. Chickens exposed to cyclic high ambient temperatures displayed lower skeletal muscle anserine and carnosine content than control chickens. Balenine could not be detected in the pectoral muscle of either group. The pectoral muscles of broiler chickens kept under cyclic high-temperature exhibited significantly lower mRNA expression of carnosine synthase 1, which synthesizes carnosine and anserine; but a significantly higher mRNA expression of carnosinase 2, which degrades carnosine and anserine. Our results suggest that heat exposure decreases pectoral imidazole dipeptide content in broiler chickens. This may be attributed to a lower expression of imidazole dipeptide-synthesizing genes, but higher levels of genes involved in their degradation.

In our previous studies, we demonstrated that the primary source of relaxin 3 (RLN3) in Japanese quail is ovarian granulosa cells. Although several relaxin family peptide (RXFP) receptors have been sequenced, the intricacies of these receptors in avian species remain insufficiently clarified. Therefore, we assessed the expression of RXFP receptors, RXFP1 and 3, in Japanese quail. Using RT-PCR, we found that both RXFP1 and 3 were ubiquitously expressed. The expression level of RXFP1 is significantly higher in the ovarian theca layer, indicating that it is the primary receptor for RLN3 in the ovary. During follicular development, there was an elevation in thecal RXFP1 expression, but it declined after the luteinizing hormone (LH) surge. We found that the protease activity of the 60 kDa band increased after the LH surge, suggesting the involvement of RLN3 signaling in ovulation. These results suggest a paracrine role of RLN3, involving its binding with RXFP1 in ovarian theca cells. This interaction may elicit biological actions, potentially initiating ovulation after the LH surge.

Numerous studies have assessed the benefits and optimal dosage of supplementation with essential oils (EOs), including extracts from plants of the Apiaceae family, as an alternative to antibiotic growth promoters (AGPs) in broilers. However, little consideration has been given to the actual chemical composition of the evaluated EOs when drawing critical conclusions, even though EO composition can vary with different extraction conditions and plant characteristics. Therefore, the present study was conducted to evaluate the effects of EOs from seeds of plants of the Apiaceae family: coriander (CEO), ajwain (AjEO), and dill (DEO), containing 56.8% linalool, 68.2% thymol, and 41.1% carvone, respectively, on the growth performance, gut health, and immune status of broilers. In total, 660 one-day-old broiler chicks were divided into 11 experimental diet groups and fed for 35 days with either the control diet, basal diet with added AGP (lincomycin, 500 mg/kg), or one of nine EO diets supplemented with CEO, AjEO, or DEO at 200, 400, and 600 mg/kg. Final body weights were improved by supplementation with not only AGP but also any EO except AjEO at 600 mg/kg; within each EO, supplementation of CEO at 400 mg/kg, AjEO at 200 mg/kg, and DEO at 200 mg/kg afforded the best growth performance. EO supplementation had beneficial effects on gut morphology, such as increased villus height in the duodenum, jejunum, and ileum, and against harmful microbiota, such as reduction of Escherichia coli and Salmonella spp. populations. Furthermore, EOs enhanced humoral immunity and improved meat quality by reducing drip loss, likely consequent to their antioxidant properties. Overall, this study presents evidence that CEO, AjEO, and DEO can each play a pivotal role in replacing AGPs, as well as providing information regarding optimal doses for broilers.

Flaxseed cake contains high levels of phenolic compounds, which have numerous biological activities, as well as a considerable amount of omega-3 fatty acids, such as α-linolenic acid, which remains after oil extraction. In this study, we examined the effects of flaxseed cake meal (FSCM) on the antioxidative status, lipid metabolism, egg fatty acid profile, and egg health index of white-egg laying hens. A total of 63 Hisex White laying hens were divided into three experimental treatment groups and fed diets containing 0, 5, or 10% FSCM from 48 to 58 weeks of age. Feeding with 5 and 10% FSCM did not significantly (p>0.05) influence total lipid, triglyceride, total cholesterol, very low-density lipoprotein-cholesterol, or low-density lipoprotein-cholesterol concentrations, or the high-/low-density lipoprotein ratio in the serum and egg yolk; however, 10% FSCM significantly (P<0.05) increased serum high-density lipoprotein. Dietary FSCM also did not affect (P>0.05) antioxidant markers in the eggs and blood plasma. Notably, dietary inclusion of FSCM significantly increased (P<0.05) total n-3 polyunsaturated fatty acids (PUFAs), α-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid levels in egg yolk, whereas the n-6:n-3 PUFAs ratio was markedly (P<0.05) decreased in a dose-dependent manner. Moreover, including 5–10% FSCM improved (P<0.05) egg health indices, with 10% being the most beneficial. Together, these findings indicated that the inclusion of up to 10% FSCM in laying hen diets improved egg yolk lipid and fatty acid profiles, as well as egg quality and nutritional and metabolic indices.

Collagen content is an important parameter affecting meat consistency. Sex differences in collagen were therefore studied in mature and juvenile Shamo chickens. The pectoral (PT), lateral iliotibial (ITL), medial part of puboischiofemoral (PIF), and lateral part of gastrocnemius (GCL) muscles were weighed, and their COL1A1 expression levels and total collagen content were analyzed. Body and muscle weights were significantly higher in males than in females of all ages. Muscle/body weight ratios were also higher in mature males than in females, but this difference was not observed in juveniles. In mature chickens, COL1A1 expression was higher in the PIF and GCL muscles; this was not the case in juvenile chicken muscles. Sex differences in collagen content were observed only in the ITLs of mature chickens. A positive correlation between muscle weight and intramuscular collagen content was found for PT and GCL, but not for ITL and PIF, muscles. These results suggest that the sex difference in intramuscular collagen content only occurs in specific muscles and that COL1A1 expression is not necessarily related to collagen content in mature chickens. Factors that determine the intramuscular collagen content likely differ by muscle type.

A low-protein (LP) diet may alleviate the environmental impact of chicken meat production by reducing nitrogen excretion and ammonia emissions. Thus, this study investigated the effect of a 15% reduced protein diet with or without amino acid (AA) supplementation on the growth performance of broiler chicks from 10 to 35 days of age and the underlying mechanism for loss of skeletal muscle mass. Thirty-six male broiler chicks were allocated to three experimental groups based on body weight: control, LP, and essential AA-supplemented LP (LP+AA). The body weight gain, feed conversion ratio, and weight of breast muscles and legs significantly decreased only in the LP group at the end of the feeding period. Plasma uric acid levels were significantly lower in the LP+AA group than those of the other groups. In the LP group, mRNA levels of microtubule-associated protein 1 light chain 3 isoform B were significantly higher in the pectoralis major, whereas those of atrogin-1, muscle RING-finger protein-1, and myoblast determination protein 1 were significantly higher in the biceps femoris compared to those in the control group. There were no significant differences in insulin-like growth factor 1 mRNA levels in the liver or skeletal muscle between groups. These findings suggested that supplementation with essential AAs ameliorated the impaired effects of an LP diet on growth performance in broiler chicks, and that the transcriptional changes in proteolytic genes in skeletal muscles might be related to the impaired effects of the LP diet.

Iris color is a prominent phenotypic feature of quail. To understand the mechanism of melanin deposition related to quail iris color, iris tissues were selected from Beijing white and Chinese yellow quail for transcriptome analysis. Differentially expressed genes (DEGs) associated with pigmentation were identified using RNA sequencing and validated by quantitative real-time polymerase chain reaction (RT-qPCR). The identified single nucleotide polymorphisms were studied using bioinformatics and iris color correlation analyses. A total of 485 DEGs were obtained, with 223 upregulated and 262 downregulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. Thirty-two genes were annotated using the GO database. Three important pigment synthesis pathways (Notch signaling, melanogenesis, and tyrosine metabolism) were identified in quail iris tissue (P < 0.05). The expression levels of solute carrier family 45 member 2 (SLC45A2), tyrosinase-related protein 1, vitamin D receptor, opsin 5, and docking protein 5 were significantly different between Beijing white and Chinese yellow quail, as verified by RT-qPCR. The c.1061C>T mutation in SLC45A2, which caused a single amino acid change at position 354 (threonine to methionine), was significantly associated with iris color in Beijing white and Chinese yellow quail, and might be the main reason for the different iris colors between these two quail species.

Lactobacillus spp. inhibit the growth of Campylobacter spp. in vitro. However, in chicken crops, in which Lactobacillus spp. predominate, such inhibition of Campylobacter has not been confirmed. In our previous study, feeding paddy rice to broiler chicks increased the residence time of the food, which might enhance the bactericidal activity of the crop. Here, the bactericidal activity against the remaining Campylobacter spp. in broiler crops was evaluated. A suspension prepared by mixing Campylobacter jejuni and titanium dioxide (TiO₂) was inoculated into the pharynx of 26-day-old broiler chicks fed a paddy rice-based diet. The crop contents were sampled at 20-min intervals. The TiO₂ residual ratio in the crop gradually decreased with time after inoculation, with 57% of the inoculated TiO₂ remaining in the crop 60 min after inoculation. The survival fraction of C. jejuni in the crops was 11% at 40 min, only 1% at 60 min, and was undetectable at 80 min. Most of the inoculated C. jejuni died in the crop before entering the next segment. These data indicated that bacterial death occurred between 30 min and 40 min after inoculation. The average survival time of C. jejuni in the crop was calculated to be 37.1 min. Thus, C. jejuni remaining in a chicken crop for more than 40 min died.

Cadaverine is a bioactive substance derived from lysine degradation by lysine decarboxylase and has gained attention for its physiological effects. Studies in rodents have revealed its role as a cell growth regulator, particularly intestinal bacterial-produced cadaverine. However, the nutritional and physiological roles of cadaverine during the embryonic period remain unclear, especially considering the immature state of the gut microbiota and digestive functions during this stage. This study explored the potential functions of cadaverine as a nutritional and metabolic signal during chicken embryonic development. Experiments were conducted using an in ovo administration method to evaluate the effects of nutritional bioactive substances on developing chicken embryos. Although there were no observable changes in body or organ weights of newly hatched chicks following in ovo cadaverine administration to day 18 chick embryos, plasma tryptophan, N^τ-methylhistidine, and N^π-methylhistidine concentrations decreased and the gene expression of insulin/insulin-like growth factor 1 signaling in skeletal muscle was upregulated. These findings imply that cadaverine influences tryptophan metabolism and skeletal muscle catabolism during the embryonic period, suggesting its role as a bioactive factor contributing to energy metabolism signaling in skeletal muscle.

Broodiness in egg-laying hens (EHs) leads to ovarian atrophy, resulting in reduced egg-laying performance. However, the ovarian regulatory mechanisms in broody hens (BCs) remain elusive. Therefore, ovaries were removed from 300-day-old BCs and EHs for RNA sequencing. Ovarian morphology and histological characteristics of the BC and EH groups were compared and analyzed. The EH group had significantly more hierarchical follicles (HFs) and small yellow follicles (SYFs) than that of the BC group. Although several secondary follicles (SFs) and primary follicles were observed in the ovaries of the EH group, only a few SFs were observed in the ovaries of the BC group. Subsequently, RNA-sequencing analysis was conducted to determine the ovarian expression profiles of the two groups. Transcriptome sequencing identified 259 differentially expressed genes (DEGs) between the BC and EH groups. Of the 259 DEGs, 136 were upregulated and 123 were downregulated. The DEGs were mapped to 22 gene ontology terms and 4 Kyoto Encyclopedia of Genes and Genomes pathways for ovarian tissue. The analysis showed that matrix metalloproteinases 11/13 (MMP11/MMP13) were enriched in the extracellular matrix. The extracellular matrix mediated by MMP13 is affected by follicle-stimulating hormone, prolactin, and estrogen, which are critical signaling pathways that may affect ovarian follicle development to regulate the large yellow follicle reserve process and the ovulation cycle of broody Chahua chickens. These findings indicate that understanding differences in gene expression between the ovarian tissues of BCs and EHs could serve as a valuable reference point for enhancing egg-laying performance in Chahua chickens.

The implementation of more animal welfare-centric livestock production systems holds considerable potential for achieving sustainable production in Japan. However, such systems generally reduce productivity per unit area, impose financial burdens on farmers that adversely affect their physical and mental health, and require increased consumer awareness. In particular, declines in productivity per unit area substantially affect the farming economy, giving rise to concerns about the sufficient egg production for the population, which consequently exacerbates Japan’s food security concerns.In this study, our goal was to predict temporal trends in the Japanese transition to welfare-centric facilities by analyzing desired egg consumption. We formulated five scenarios, ending in 2050, to assess the future impact of changing the proportions of types of egg production on total production, numbers of hens, total farm area, production costs, and retail prices. Multiple issues were identified in our analysis. First, Japanese production uses conventional caging, which provides high efficiency, maximum output, and decreased prices. Our simulations suggest that ensuring optimal production is likely to present challenges as animal welfare is addressed. They also suggest that the poultry industry or the Japanese government will likely need to plan changes in production methods will likely be necessary to prevent egg surpluses and/or shortages. Additional issues were identified, including price increases, numbers of hens, total farm area, and production costs. This study offers valuable insights for policymakers in their decision-making processes to help optimize production methods while considering food security, animal welfare, and farmers’ welfare.

Helmeted guinea fowl are social animals and only males form a hierarchy in the wild. Non-cage husbandry systems benefit the reproductive health of guinea fowl; however, there are concerns that the feeding duration of subordinate individuals is insufficient. Here, the pecking orders formed during small-scale floor feeding were investigated. There were three experimental categories: male-only (four males), female-only (four females), and mixed category (two males, two females). Each experimental category was set up three times and included different individuals. Behaviors were recorded for 130 h 52 min, 89 h 11 min, and 98 h 46 min in the male, female, and mixed categories, respectively. Male helmeted guinea fowls pecked other males, whereas females exhibited little pecking behavior. Male pecking behavior was not homogeneous within each experimental group. It has been suggested that males form a pecking order, whereas females have no hierarchy under small-scale floor-feeding conditions, as observed in the wild. In most cases, on the first day the number of pecking behaviors was low 20 min after the start of the experiment. The three subordinate individuals in the mixed category had little time to feed, whereas the other birds in the mixed category and all helmeted guinea fowl in the male- and female-only categories had longer feeding durations. We suggest that helmeted guinea fowl may be reared under small-scale floor feeding, and that the health of males should be managed. However, rearing females and males under small-scale floor feeding conditions should be avoided. This study contributes to improving the welfare of helmeted guinea fowl reared under small-scale floor feeding.

This study evaluated the effects of whole flaxseed (WFS), fish oil (FO), and different sources of Se, Zn, and Fe (inorganic, organic, and nano-source) on egg production, quality, sensory attributes, and serum protein metabolites in laying hens. A total of 144 hens were divided into six groups with six replicates of four hens each. Hens were fed six diets as follows: 1) control diet; 2) 7.5%WFS+1.5%FO; 3) 7.5%WFS+1.5%FO+175 mg/kg vitamin E (VE); 4) 7.5%WFS+1.5%FO+175 mg/kg VE + inorganic sources of Se, Zn, and Fe (ISeZnFe); 5) 7.5%WFS+1.5%FO+175 mg/kg VE + organic sources of Se, Zn, and Fe (OSeZnFe); 6) 7.5%WFS+1.5%FO+175 mg/kg VE + nano-source of Se, Zn, and Fe (NSeZnFe) from 40–50 weeks of age. Laying hens fed 7.5% WFS, 1.5% FO, and different sources of trace elements in their diets had no negative effects on laying rate, egg weight, egg mass, feed intake, feed conversion ratio, body weight change, or survival rate compared to that of hens fed the control diet. Dietary treatments did not negatively affect the external and internal egg characteristics or egg sensory attributes. Feeding 7.5%WFS+1.5%FO+VE+ISeZnFe positively influenced yolk color in fresh eggs. Dietary treatments had a significant impact on egg nutritional composition, with the highest levels of macronutrients found in eggs from hens fed the 7.5%WFS+1.5%FO+VE+NSeZnFe treatment. The highest plasma globulin concentrations were observed in hens fed organic and nano-source trace elements. The same diets reduced plasma uric acid levels. Based on these findings, the inclusion of organic or nano-source trace minerals in diets containing WFS and FO positively affected egg quality and hen protein metabolites.

Vaccination is important for reducing disease incidence in the poultry industry. To enhance immunity and vaccine efficacy, chicken cytokines associated with antibody production must be identified. In this study, we focused on interleukin-5 (IL-5), involved in antibody production in mice, measuring its expression and effects on antibody production. Concanavalin A-stimulated splenocytes were used for RT-PCR to clone IL5 cDNAs. Recombinant IL-5 was prepared from the clone and administered to chickens with antigen via the ocular-topical route twice every alternate week. IL-5 enhanced antigen-specific IgY and inhibited antigen-specific serum IgA production in serum. Our findings suggest that IL-5 plays an important role in chicken antibody production, with possible unique functions.

In this study, we examined whether brown rice and sake lees (domestic feed ingredients) could replace corn and soybean meal (major imported feed ingredients) in broiler chick feed. In Experiment 1, 21-day-old broiler chicks were assigned to two groups and fed a corn-soybean- or a brown rice-soybean-based diet for three weeks (3 birds × 4 replicates/group). Dietary brown rice significantly improved body weight gain and feed conversion ratio. Brown rice feeding also significantly increased L* (lightness) in the thigh and significantly decreased a* (redness) and b* (yellowness) in the thigh and b* in the fat. In Experiment 2, 21-day-old broiler chicks were assigned to three groups and fed either a corn-soybean-based diet for 3 weeks, a corn-soybean-based diet for the first 2 weeks followed by a brown rice sake lees-based diet for the last week, or a brown rice sake lees-based diet for 3 weeks (3 birds × 4 replicates/group). Replacement of the imported feed ingredients significantly improved the feed conversion ratio. The a* values for the breast, thigh, and fat, and the b* values for the thigh and fat were significantly decreased by rice and sake lees feeding for 3 weeks. The a* values for the breasts and fat were significantly decreased by rice and sake lees feeding for 1 week. These results suggest that brown rice and sake lees can be used as replacements for imported feed ingredients such as corn and soybean meal in broiler chicks without detrimental effects on growth performance. These domestic feed ingredients may benefit local production and consumption of poultry in Japan.

Wooden breast myopathy and leg weakness are serious problems in the broiler chicken industry. The color and intensity of light in the chicken habitat affect behavior, including walking of chicks. The present study was conducted to determine whether periodic and local light switching induces locomotion and affects wooden breast myopathy and leg weakness in broiler chicks. Thirty five-day-old broiler chicks were assigned to two pens (4.72 m × 0.73 m each). In the control pen, chicks were reared under three white-light emitting diode (LED) lights until they were 42 days old. In the other pen, chicks were reared under a white LED light located in the center, supplemented with blue or red LED lights on either side of the pen. The color of the LED lights changed every 3 h, from blue and red to red and blue. From 21 d of age, all LED lights were changed and only one of the side lights was turned on every 3 h. From 35 d of age, all three white lights were turned on until 42 d of age. Periodic and local color switching and on-off switching significantly induced locomotion in broiler chicks. Wooden breast scores tended to improve with light-switching treatment. The tibia length, diameter, and breaking strength were not significantly affected. This is the first report showing that locomotion may be induced in broiler chicks by periodic and local lighting switching, and may be useful for improving the health status of broiler chicks.

This study investigated the distribution of ofloxacin-resistant Escherichia coli (OFXR-EC) in broiler parent flocks (PS) treated with ofloxacin for 5 days from the time the chicks arrived at the poultry house, and their broiler offspring. OFXR-EC was detected in 22.95% of neonatal parent stock chicks (PSN) arriving at the poultry house. The detection rate of OFXR-EC in PS rearing was 72.49%, which was significantly higher than that detected in PSN. In addition, the detection rate of OFXR-EC was significantly lower in neonatal chicks of their offspring broilers (CSN) at 7.06% than that of PS, but was 24.62% in offspring broiler flocks (CS) at approximately 6 weeks of age. The OFXR-EC detection rate was significantly higher in CS than that in CSN, even though no therapeutic antimicrobials, including ofloxacin, were used from CSN to CS. In addition, the proportions of OFXR-ECs in E. coli isolated from samples in which OFXR-ECs were detected were 63.85% for PSN, 10.52% for PS, 62.00% for CSN, and 8.25% for CS. There was little difference in the composition ratio of OFXR-EC between PSN and CSN, or between PS and CS.