Hongyan Kou’s research while affiliated with Zhongkai University of Agriculture and Engineering and other places

Zinc plays a crucial role in the antioxidant capacity, and inflammatory response of aquatic species, but its impact on largemouth bass Micropterus salmoides is rarely reported. Therefore, this paper aimed to investigate the effects of different levels of zinc on the growth performance, histopathology, antioxidant capacity, and inflammatory cytokines of largemouth bass Micropterus salmoides. Fish with an initial weight of 7.84 ± 0.06 g were cultured for 10 weeks. Five experimental diets were prepared with supplemented proteinate Zn (Bioplex Zn, Alltech) (0, 30, 60, 90, and 120 mg/kg), which were named the Zn-42, Zn-73, Zn-103, Zn-133, and Zn-164 groups. No evident difference was found between the dietary zinc level and the survival rate, the crude lipid content of the whole fish, or the visceral somatic index. Weight gain, condition factor, whole-body crude protein content, interleukin-10, and transforming growth factor beta gene expression were gradually enhanced with up to 102.68 mg/kg zinc and decreased at higher levels. The hepatosomatic index, feed conversion ratio, malondialdehyde level in the liver, aspartate aminotransferase, and alanine transaminase activity in the serum, gradually decreased up to 102.68 mg/kg zinc, and gradually increased beyond this. Activation of the nuclear factor erythroid-derived 2-like 2/Kelch-like ECH-associated protein 1 signaling pathway gradually up-regulated the mRNA levels and activities of glutathione peroxidase, total antioxidant capacity, catalase, and superoxide dismutase in the liver, this antioxidant ability was lower when the zinc was greater than 102.68 mg/kg. The gene expressions of nuclear factor-k-gene binding and pro-inflammation cytokines (interleukin-1β, interleukin-15, tumor necrosis factor alpha, and interleukin-8) were up-regulated up to 102.68 mg/kg zinc and then gradually repressed. In conclusion, using broken line analysis to estimate weight gain and Zn proteinate as the zinc source, the recommended dietary zinc for largemouth bass is 66.57 mg/kg zinc.

Toll-like receptors 5 (TLR5), a member of the toll-like receptors (TLRs) family, is a class of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). It responds to vertebrate recognition of bacterial flagellin and participates in innate immune responses. However, genome-wide identification and characterization of TLR5 in fishes have not been investigated. Here, three TLR5M isotypes (TLR5Ma, TLR5Mb1, and TLR5Mb2) and a TLR5S are all extracted from fish genomes on the basis of phylogenetic and synteny analyses. We confirmed that the non-teleost fishes have one TLR5M gene, as well as additional TLR5 genes (TLR5M and TLR5S) in teleost fishes. In addition, some special teleost fishes possess two to three TLR5 genes, which have undergone the fourth whole-genome duplication (WGD). According to our results, we inferred that the diversity of TLR5 genes in fishes seems to be the result of combinations of WGD and gene loss. Furthermore, TLR5 isoforms displayed differences at the flagellin interaction sites and viral binding sites, and showed lineage-specific, which indicated that TLR5 duplicates may generate functional divergence. Bacterial experiments also supported the idea that CiTLR5Ma and CiTLR5Mb are subfunctionalized to sense bacterial flagellin. In summary, our present comparative genomic survey will benefit for further functional investigations of TLR5 genes in fish.

An 8-week feeding trial was performed to assess the influence of a gradient of protein levels (14.38–45.23%) on flesh quality, skin color, amino acid profile, collagen, antioxidant capability, and antioxidant-related signaling molecule expression of the softshell turtle (Pelodiscus sinensis). Hardness, gumminess, chewiness, and yellowness values in the plastron and carapace, along with collagen, superoxide dismutase, catalase, total antioxidant capacity, and glutathione peroxidase, all improved with elevating dietary protein up to 26.19%, after which they leveled off. Additionally, total amino acids, flavor amino acids, essential amino acids, and non-essential amino acids in the muscle, as well as the expression of copper/zinc superoxide dismutase, glutathione peroxidase, catalase, manganese superoxide dismutase, NF-E2-related factor 2 were all enhanced by increasing the dietary protein level but not changed by higher protein levels. When dietary protein levels were less than 26.19%, the mRNA expression of Kelch-like ECH-associated protein 1, malondialdehyde, and redness values in the carapace and plastron were reduced, as was the lightness values of the carapace, all of which plateaued at higher protein levels. Using catalase activity and malondialdehyde as the indicators and applying a broken-line analysis, the optimal dietary protein level for P. sinensis was inferred to be 26.07 and 26.06% protein, respectively. In summary, an optimal protein input improved turtle flesh quality by strengthening antioxidant capacity in muscle tissue and by regulating the expression of antioxidant-related enzymes via the Nrf2/keap1 signaling pathway.

In aquatic animals, dietary protein plays a crucial role in their growth and immunity. A feeding trial was conducted on soft-shelled turtles (Pelodiscus sinensis) to assess the effects of various levels of protein on the specific growth rate (SGR), ambient water quality (total ammonia nitrogen (TAN), total nitrogen (TN) and total phosphorus (TP)), hematological parameters (respiratory burst (RB), red blood cell count (RBC), albumin content (Alb), hemoglobin level (Hb) and osmolality), plasma immunoglobulin M (IgM) levels and lysozyme activity. Soft-shelled turtles weighing about 4.02 g were fed fish meal-based diets with 14.38%, 20.41%, 26.19%, 32.23%, 37.63% and 45.23% protein for 8 weeks. SGR, RBC, Hb, Alb, RB, IgM and lysozyme activity were enhanced as the dietary protein was increased from 14.38% to 26.19%, then reached a plateau. For identical feeding times, TAN and TN were increased with elevating dietary protein levels. While, no statistically significant differences were observed among the 26.19%, 32.23% and 37.63% groups. When the turtles were cultivated for 56 days and fed with 45.23% protein, the TP in the culturing water was higher than that in the other groups. An increase in dietary protein level up to 26.19% increased the RNA/DNA ratio, which subsequently plateaued at a steady level. The levels of dietary protein had no impact on osmolality or alkaline phosphatase (AKP) activity. On the basis of broken-line analyses derived from SGR, the optimum dietary protein level for soft-shelled turtles was found to be 27.11% protein.

A 12-week growth trial was conducted to investigate the impacts of dietary zinc on growth performance, whole-body composition, haematological parameters, liver aspartate aminotransferase and alanine transaminase activity and tissue trace mineral contents of soft-shelled turtle (Pelodiscus sinensis). Approximately 4 g of turtles were fed fishmeal-based diets with 35.43, 46.23, 55.38, 66.74, 75.06 and 85.24 mg Zn/kg. Respiratory burst, red blood cells and intestine somatic index enhanced with escalating dietary Zn concentrations up to 55.38 mg/kg and then kept constant beyond this level. Escalating dietary Zn inclusion up to 66.74 mg/kg improved weight gain, survival rate, feed intake, albumin, haemoglobin, liver aspartate aminotransferase and alanine transaminase activity, carapace ratio and crude protein of turtles, beyond which they decreased. Osmolality, hepatosomatic index and crude lipid of turtles firstly declined with elevating dietary zinc levels from 35.43 to 55.38 mg/kg diet and then increased at a higher zinc level. Liver and muscle Zn concentrations enhanced with escalating dietary zinc inclusion, whereas Cu and Fe concentrations reduced. In summary, the optimal dietary zinc requirements for turtles were 61.27, 60.46 and 60.03 mg/kg diet, based on quadratic regression analysis derived from weight gain, aspartate aminotransferase and alanine transaminase activity, respectively.

Zinc (Zn) plays a role in the antioxidant capacity and immunity of aquatic animals. A twelve-week feeding experiment was performed to estimate the impact of dietary zinc on antioxidant enzyme-related gene expression, antioxidant enzyme activity and non-specific immune functions of soft-shelled turtles, Pelodiscus sinensis. Six fishmeal-based experimental diets with 32.45% protein were formulated, which contained 35.43, 46.23, 55.38, 66.74, 75.06 and 85.24 mg/kg Zn, respectively. Catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels improved with an elevation in dietary Zn from 35.43 to 55.38 mg/kg and then reduced when dietary Zn was further elevated. The expression levels of Nrf2 and antioxidant-related genes CuZnSOD, MnSOD, CAT, GPX1, GPX2, GPX3 and GPX4 escalated with elevating Zn concentration up to 55.38 mg/kg in diets and then reduced as dietary Zn elevated. The expression levels of Kelch-like ECH-associating protein 1 (keap1) showed a reverse trend with that of Nrf2. The contents of malondialdehyde (MDA) in the 55.38 and 66.74 mg/kg Zn diet-fed groups were the lowest. Alkaline phosphatase activity (AKP), superoxide anion (O2⁻), lysozyme activity and total antioxidant capacity (T-AOC) improved with an escalation in dietary Zn concentration up to 66.74 mg/kg. Optimal dietary Zn improved antioxidant capability, immunity, and antioxidant enzyme-related gene expression. The dietary Zn demand for soft-shelled turtles were 60.93 and 61.63 mg/kg, based on second regression analysis of SOD and T-AOC activity, respectively.

The impacts of dietary protein on growth, body proximate composition, feed utilization, amino acid profile, digestive enzyme activity and amino acid metabolizing enzymes in soft‐shelled turtles (Pelodiscus sinensis) (initial weight: 4.02 ± 0.06 g) were measured during an 8‐week feeding trail. Six diets containing 8%, 18%, 28%, 38%, 48% and 60% (control group) fishmeal were formulated, which contained 14.38%, 20.41%, 26.19%, 32.23%, 37.63% and 45.23% protein respectively. Weight gain, mean feed intake, skirt ratio, pepsin activity of stomach, total amino acid of the muscle and crude protein levels of the whole body improved as dietary protein content increased from 14.38% to 26.19% but remained stable thereafter. The hepatosomatic index, feed conversion ratio, intestinal alpha‐amylase activity, moisture content of the whole body and serum alanine transaminase and aspartate aminotransferase activity decreased with increasing protein content up to 26.19% and then plateaued. The protein productive value and protein efficiency ratio decreased as protein content increased. Intestinal lipase activity and crude lipid levels of the whole body were unaffected by dietary protein level. Hepatic alanine transaminase and aspartate aminotransferase activity first significantly increased with increasing protein but remained stable at protein content ≥26.19%. The optimum dietary protein level for soft‐shelled turtles was in the range of 27.36%–28.46% based on weight gain, ALT activity and AST activity results.

Hawthorn fruit has long been used as a functional food in China due to its effects of increasing appetite, promoting digestion and protecting the gastrointestinal tract. The present study was conducted to assess the impact of dietary hawthorn extract (HTE) supplementation on plasma biochemical indices, proximate body composition, skin coloration, intestinal morphology and changes in mRNA expression of immune-related genes and tight junction proteins genes in the intestine of golden pompano. Basal diets supplemented with HTE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg⁻¹ were fed to golden pompano for eight weeks. The study showed dietary HTE supplementation had hypolipidemic effects by reducing plasma cholesterol, triglyceride and low density lipoprotein content and improved skin coloration by increasing yellowness values of the ventral skin color of golden pompano. Meanwhile, dietary supplementation of 0.50 and 1.00 g kg⁻¹ HTE could improve gut morphology by increasing villus length, villus width and muscle thickness, and enhance barrier function by increasing tight junction protein genes mRNA levels. However, dietary 10.00 g kg⁻¹ HTE supplementation not only caused damage to intestinal morphology and barrier function, but also induced oxidative stress and inflammatory response. Our study demonstrated that dietary 0.50 and 1.00 g kg⁻¹ HTE supplementation was beneficial for skin coloration and intestinal health of golden pompano.

Intermuscular bones (IBs) are only found in the muscles of fish. Bone morphogenetic protein 2 (bmp2) is considered to be the most active single osteogenesis factor. It promotes cell proliferation and differentiation during bone repair, as well as inducing the formation of bones and cartilages in vivo. However, detailed investigations of this family in fish are incredibly limited. Here, we have used a variety of published and unpublished bmp2 sequences for teleosts and cartilage fish in order to explore and expand our understanding of bmp2 genes in fish. Our results confirmed that teleost genomes contain two or more bmp2 genes, and the diversity of bmp2 genes in vertebrates appears to be as a result of a combination of whole genome duplication (WGD) and gene loss. Differences were also observed in tissue distribution and relative transcription abundance of the bmp2s through a transcriptomic analysis. Our data also indicated that bmp2b may play an important role in the formation of IBs in teleosts. In addition, protein sequence alignments and 3D structural predictions of bmp2a and bmp2b supported their similar roles in fishes. To summarize, our existing work provided novel insights into the bmp2 family genes in fishes through a mixture of comparative genomic and transcriptomic analysis.