Data on plasma cortisol levels in nibbler fish Girella punctata reared under high-density conditions in either surface seawater or deep ocean water.

Deep ocean water (DOW) is the water obtained from depth of >200 m below the surface of Earth's oceans and is characterized by rich nutrients and cleanliness [1,2]. We have recently reported that DOW suppresses the high-density-induced increase of plasma cortisol levels (i.e., a stress marker) in Japanese flounder (Paralichthys olivaceus) [1]. The current study aimed to examine whether the cortisol-reducing effect of DOW was observed in other marine organisms as well by comparing the plasma cortisol levels of nibbler fish Girella punctata reared under high-density conditions between surface seawater (SSW) and DOW. The nibbler fish were caught from Tsukumo Bay of Noto Peninsula (Ishikawa Prefecture, Japan). The DOW was obtained from seawater 320 m below the Noto Bay surface at a facility (Aquas Noto, Ishikawa Prefecture, Japan), whereas SSW was obtained from Tsukumo Bay (Noto Peninsula, Ishikawa Prefecture). The dissolved oxygen was maintained at approximately 7 mg/L in DOW as well as in SSW. Before they were transferred to the high-density condition, nibbler fish were acclimated in SSW at 20°C for 1 week at a mean density of 100 g/62.5 L. To expose them to the high-density stress, each of fish was kept at a density of 10 kg/m3 in a single aquarium (60 × 25 × 30 cm) containing either SSW or DOW (n = 8). Subsequently, the fish were reared with SSW or DOW for 10 days at 20°C ± 1°C under a 12:12-h light-dark cycle. A heparin containing syringe was used to obtain the blood samples from the caudal vessels of the fish anesthetized with a 0.04% 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation). The blood sampling was performed on days 0, 5, and 10 after rearing in the small aquaria. The plasma samples were prepared from the collected blood by centrifuging it at 5200 × g for 5 min and the cortisol concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cosmo Bio Co. Ltd., Tokyo, Japan) from those samples. The plasma cortisol concentration of nibbler fish reared in SSW on day 10 was significantly higher than that on day 0, whereas those reared in DOW did not show significant difference on the respective days. The current data contributes to the generalization of the cortisol-reducing effect of DOW on fish, which has been proposed in Japanese flounder [1]. These data could be used for developing and designing experiments to analyze the mechanisms underlying the cortisol-reducing effects by using small fish such as zebrafish, a well-established animal model.

Molecular characterization of TNF-ß and IFN-γ in yellowfin seabream (Acanthopagrus latus, Hottuyn, 1782) and their immune responses to density stress during transport.

The inflammatory cytokines TNF-ß and IFN-γ are important mediators of the vertebrate inflammatory response and coordinators of the immune system in regard to NF-κB signalling pathways. In this study, the TNF-ß and IFN-γ genes of yellowfin seabream, Acanthopagrus latus were identified, and the multiple sequence alignments, evolutionary relationships and gene expressions of the two genes were also determined. AlTNF-ß contained a 762 bp open reading frame (ORF) encoding 253 amino acids, while AlIFN-γ contained a 582 bp ORF encoding 193 amino acids. An amino-acid sequence alignment analysis showed that these proteins have highly conserved transmembrane structural domains among teleosts. Moreover, AlTNF-ß has a close affinity with TNF-ß of yellowfin seabream while AlIFN-γ has a high evolutionary correlation with A. regius and Sparus aurata. In addition, the mRNAs of AlTNF-ß and AlIFN-γ are widely expressed in various tissues. AlTNF-ß is highly expressed in gill and intestinal tissues, and the mRNA levels of AlIFN-γ are higher in spleen, skin, and gill tissues than in other tissues. Under transportation density stress, the mRNA level of AlTNF-ß was significantly elevated in the intestine of the high-density group, while AlTNF-ß transcription in the gills did not vary significantly among the density groups. Furthermore, AlIFN-γ expression was increased in liver, intestinal, and gill tissues under high transportation density. The results of this study show that TNF-ß and IFN-γ expression in yellowfin seabream is greatly affected by density stress. The density of 125 per bag for 4-5 cm fry or 1200 per bag for 1-2 cm fry is most suitable for the transportation of live fish. These results might provide a reference for further studies on the immunomodulatory response process and auxiliary function of immune stress of TNF and IFN genes in fish under density stress.

Effects of dietary chlorogenic acid on ileal intestinal morphology, barrier function, immune factors and gut microbiota of broilers under high stocking density stress.

Aims: The purpose of this research was to assess the effect of chlorogenic acid (CGA) in the diet on ileac structure, barrier function, immunological state, and microbial profile of broiler chickens in a high stocking density (HD) environment. Methods: Four hundred and seventy-six male AA broiler chickens were randomly split into four groups, two with a normal stocking density (ND) of fourteen birds per m2 and two with a high stocking density of twenty-two birds per m2. Each of the treatments consisted of five replicates. One of the two ND and HD groups received the usual feed, while the other two were given at 1.5 g/kg CGA as part of their dietary regimen. Results: The ND CGA group showed a greater increase in villus height and villus height/crypt depth compared to the ND group at 35 and 42 days. The HD group experienced a greater elevation in villus height due to CGA supplementation than the HD group across days 28, 35, and 42. At day 42, the HD group saw a decline in OCLN and ZO-1 mRNA expression in the ileum, but CGA was able to restore them. The HD group experienced a greater rise in OCLN mRNA than the control HD group when supplemented with CGA. The expression of TNF-α, IL-1ß, and IL-6 in the ileum was higher in the HD group, and CGA supplementation enhanced this effect. The HD group experienced a greater rise in IL-10 mRNA expression than the control group following the administration of CGA. The HD group showed reduced alpha diversity and an increase in detrimental microbes such as Turicibacter and Shigella in the gut compared to the ND group, while the HD CGA group saw a reduction in Turicibacter, Shigella, and other harmful microbes. These findings reveal that HD stress suppressed the growth of ileac villi, decreased the expression of tight-junction genes, amplified the expression of inflammatory genes, and disturbed the gut microbiota, ultimately leading to increased intestinal permeability. Conclusion: We conclude that when chickens are given dietary CGA, the disruption of the ileac barrier and increased oxidative damage and inflammation due to HD stress are reduced, which increases ileac integrity and the presence of beneficial intestinal bacteria.

Dietary Supplementation with Chlorogenic Acid Enhances Antioxidant Capacity, Which Promotes Growth, Jejunum Barrier Function, and Cecum Microbiota in Broilers under High Stocking Density Stress.

Chlorogenic acids (CGA) are widely used as feed additives for their ability to improve growth performance and intestinal health in poultry. However, whether dietary CGAs could reverse the impaired intestinal condition caused by high stocking density (HD) in broiler chickens is unknown. We determined the effect of dietary CGA on growth, serum antioxidant levels, jejunum barrier function, and the microbial community in the cecum of broilers raised under normal (ND) or HD conditions. HD stress significantly decreased growth and body weight, which was restored by CGA. The HD group showed increased serum malondialdehyde, an oxidative byproduct, and decreased SOD and GSH-Px activity. CGA reduced malondialdehyde and restored antioxidant enzyme activity. HD stress also significantly decreased jejunal villus length and increased crypt depth. Compared with ND, the expression of tight-junction genes was significantly decreased in the HD group, but this decrease was reversed by CGA. HD also significantly upregulated TNF-α. Compared with ND, the cecal microbiota in the HD group showed lower alpha diversity with increases in the harmful bacteria Turicibacter and Shigella. This change was altered in the HD + CGA group, with enrichment of Blautia, Akkermansia, and other beneficial bacteria. These results demonstrated that HD stress decreased serum antioxidant capacity, inhibited the development of jejunal villi, and downregulated expression of tight-junction genes, which increased intestinal permeability during the rapid growth period (21 to 35 days). Dietary CGA enhanced antioxidant capacity, improved intestinal integrity, and enhanced beneficial gut bacteria in chickens raised under HD conditions.

Effect of stocking density and vitamin E or zinc supplementation on growth, physiology, gene expression, and economic efficiency of growing broiler chicks.

A total of 636 1-day-old male Cobb chicks were randomly assigned to seven treatments. The chicks were offered feed and water ad libitum throughout the experimental period. The first three groups included different stocking densities of broiler birds (low stocking density, LSD: 23 kg/m2; medium stocking density, MSD: 34 kg birds/m2; and high stocking density HSD: 39 kg birds/m2). The LSD group was considered a control group. The other four groups included MSD or HSD broiler birds supplemented with either Vit E (100 mg/kg DM diet; MSDVE and HSDVE) or Zn (100 mg/kg DM diet; MSDZn and HSDZn) in their basal diet. The main findings indicated that HSD and MSD negatively affected (p < 0.05) all variables under investigation compared with LSD. Compared with LSD, broiler birds in the MSD and HSD groups had lower body weights and higher feed conversion ratios, higher concentrations of blood plasma hormones (triiodothyronine thyroxine and corticosterone), and downregulated expression levels of hepatic growth hormone and insulin-like growth factor-l. In addition, broiler birds stocked at medium or high densities resulted in less economic return and profit. Vit E or Zn supplementation to broiler birds stocked at medium or high densities significantly reversed all adverse effects of HSD (> 23 kg/m2) on growth performance, hormones, and gene expression. It could be recommended that adding Zn at a level of 100 mg/kg per DM diet allows increasing the stocking density of broiler birds from 23 kg/m2 to 34 birds/m2 while maintaining the birds, welfare and economic profit.

[Elucidation of Phenomena Involving Cyanobacteria in Freshwater Ecosystem by Chemically Ecological Approach].

Lakes Sagami and Tsukui are reservoirs constructed by connecting to the Sagami River. Because of eutrophication of the lakes, cyanobacteria have appeared every year. This review deals with phenomena related to occurrence of cyanobacteria that have been observed for 40 years since 1974 at the lakes. These 40 years of observations raised three interesting issues including the retention of cyanobacteria on their surfaces. These phenomena have been attributed to the usual factors, such as illuminance, nutrition and water temperature, but our research results suggested that they cannot be resolved without the introduction of another factor. We have attempted to elucidate various phenomena involving cyanobacteria in lake ecosystems by chemical ecological methods using volatile organic compounds (VOCs) produced by the cyanobacteria as indicators. One of the VOCs, ß-cyclocitral, was significantly involved in the above phenomena, which was considered to be produced by the carotenoid cleavage dioxygenase (CCD) of the cyanobacteria. ß-Cyclocitral was not produced in the two known CCDs, but two additional CCDs to Microcystis aeruginosa participated to produce the ß-cyclocitral. These CCDs did not directly produce ß-cyclocitral, but it was accumulated in cells as their precursors. The released ß-cyclocitral underwent a Baeyer-Villiger-like oxidation. It was speculated that Microcystis activated the CCD genes through density stress and produced ß-cyclocitral, which acted as an allelopathic substance. As a result, the number of cells of cyanobacteria decreased, and the resulting nitrogen and phosphorus were fed to the living cyanobacteria. It is postulated that this "quorum sensing" was functioning in the above-mentioned issues.

Male density, a signal for population self-regulation in Alligator sinensis.

The regulation of population density is suggested to be indirect and occurs with a time-lag effect, as well as being female centred. Herein, we present a quantitative analysis on the precise, timely and male-dominated self-regulation of Chinese alligator ( Alligator sinensis) populations. Analysis of 31 years of data revealed gender differences in regulation patterns. Population dynamics were restricted by male density rather than population density, and population growth was halted (birth rate = 0) when male density exceeded 83.14 individuals per hectare, until some males were removed, especially adult males. This rapid and accurate response supports the notions of intrinsic mechanisms and population-wide regulation response. Furthermore, density stress affected mating success rather than parental care to juveniles, i.e. females avoided unnecessary reproduction costs, which may represent an evolutionary advantage. Our findings highlighted the importance of further studies on related physiological mechanisms that focus on four characteristics: quantity breeds quality, gender differences, male density thresholds and nonlinearity.