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Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78-95.06% viability after 1-6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3-24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Grass carp (Ctenopharyngodon idellus) is one of the most essential fishing species in China. The bait for this fish is rapidly developing. However, the study on the attractants in the bait for this fish lacks. This study was designed to systematically investigate the effects of 16 kinds of test substances on the perspective of behaviour and physiology of grass carp by using different kinds of methods, including behavioral tests (maze test and biting-balls test) and electro-olfactogram (EOG). Our experiment's idea is mainly to imitate: in addition to vision, fish in nature also use smell to find food and finally swallow under the action of olfaction, taste, and other sensory systems. Firstly, the behavioral maze test was used to screen the attractive or suppressive effect of 16 test substances on grass carp, and the electronic olfactory recording method was used to further evaluate the olfactory response of grass carp to the eight stimuli selected from the maze test. Then, the best concentrations of these eight stimuli and their combination were investigated by the biting-balls test to compound a formula with the strongest appetite for grass carp. The results of behavioral maze test showed that dimethyl-β-propiothetin (DMPT), dimethylthetin (DMT), glycine, taurine, L-glutamic, L-alanine, L-proline, and L-arginine have different degrees of usefulness in attracting grass carp. The electro-olfactogram recoding showed that the EOG response of grass carp to the stimuli is a transient biphasic potential change and all of the eight stimuli could induce the EOG response of grass carp. The biting-balls test showed that glycine, L-glutamic, and L-arginine at 10 -2  mol/L had significant feeding stimulation and DMT at 10 -1  mol/L had significant feeding stimulation than the other groups. Finally, formula 9 composed of DMT, glycine, L-glutamic acid, and L-arginine has the greatest attraction for grass carp. The results of this study verified the attractive effect of some amino acids and other chemicals on grass carp fishing, and would provide support for the production of specific grass carp attractants., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The present study was conducted to investigate the regulative function of FGF6 in the muscle growth of grass carp (Ctenopharyngodon idellus) by the bioinformatics analysis and expression pattern analyses of FGF6 genes in different developmental stages and tissues, as well as the correlation analysis between muscle growth and FGF6 expression after fish were fed with different levels of dietary lotus leaf flavonoids (LLF) (0, 0.03%, 0.06%, 0.09%). Results showed that the FGF6a and FGF6b genes are two homologs of the FGF6 family, encoding 205 and 209 amino acids, respectively. Alignment of amino acid sequences and phylogenetic analysis demonstrated that FGF6a and FGF6b are highly conserved with other vertebrates. Quantitative RT-PCR analysis showed both FGF6a and FGF6b expressions were high in brain and muscle but low in other examined tissues. During embryonic development, FGF6a and FGF6b mRNA expressions could be detected as early as at fertilized egg stage and displayed the highest value at cleavage stage. Dietary LLF affected the gene expression of FGF6 in white muscle. The relative expression of FGF6a of 0.06% LLF group was significantly higher than that of 0.09% LLF group, while FGF6b expression of 0.06% LLF group was higher than those of other groups (P < 0.05). The muscle fiber diameter was significantly higher in 0.06% LLF group in comparison with other groups, while the fiber density in this group was lower (P < 0.05). Both FGF6a and FGF6b expressions were positively correlated with fiber diameter but negatively correlated with fiber density. These results collectively suggest that FGF6a and FGF6b play an important role in muscle growth regulation in grass carp.

Grass carp (Ctenopharyngodon idellus) is one of the most important aquaculture fish in China. This study tried to explore the effects of dietary alcoholic extract of lotus leaf (AELL) addition on the growth performance and health status of grass carp by feeding juvenile fish (average weight: 34 ± 1 g) with four different experimental diets: control, AELL7, AELL14 and AELL21 for 8 weeks. At the end of the growth trial, the highest values of final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR) and feed intake (FI) all occurred in group AELL14 (P < 0.05). Compared to control, the crude lipid content of whole-body and the serum malondialdehyde (MDA) in the three experimental groups decreased, while the serum superoxide dismutase (SOD), glutathione peroxidase (GSH) and total antioxidant capacity (T-AOC) values almost all increased in the three experimental groups. The highest serum immunoglobulin M (IgM) concentration occurred in AELL14 group (P < 0.05). In AELL14 and AELL21 groups, both the serum complement 3 (C3) concentration and lysozyme (LYS) activity were significantly higher, whereas the final cumulative mortality in challenge test was significantly lower, when compared to those in control group (P < 0.05). The AELL exerted dose-dependent beneficial effects on grass carp health through up-regulating related gene expressions and enzyme activity. In conclusion, the optimal dietary AELL level is 0.14% for juvenile grass carp., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Dietary arginine (Arg) could improve the intestinal structure and absorption of grass carp (Ctenopharyngodon idellus); however, the mechanism of Arg on intestinal morphology improvement was unclear. The present study aimed to explain the possible mechanism of the positive effect of Arg on intestinal epithelial cells of grass carp. An in vitro study was conducted through a primary culture model to assess the growth, cell viability, mRNA expressions of TOR signal pathway, and tight junction proteins of enterocytes after culture in the medium with 6 levels of Arg (0, 0.1, 0.2, 0.5, 1.0, and 2.0 mmol/L). The results showed that 0.5 mmol/L Arg improved the cell number and decreased the lactate dehydrogenase and creatine kinase activities in culture medium (P < 0.05). The alkaline phosphatase activity in cell lysis buffer was depressed by 1 and 2 mmol/L Arg (P < 0.05). The nitric oxide (NO) content showed an increasing trend with the Arg content (P < 0.05), whereas the NO synthase activity showed an opposite trend to NO. TOR expression was higher in 0.2 and 0.5 mmol/L groups, whereas S6K1 expression in 1.0 mmol/L and 2.0 mmol/L groups were lower (P < 0.05). The mRNA expressions of occludin, claudin 3, and claudin c in 0.5 mmol/L group were the highest, while ZO-1 and claudin b expressions were higher in 0.2 and 0.5 mmol/L groups (P < 0.05). This study indicated that Arg enhanced the growth and integrity of intestinal epithelial cells of grass carp through upregulation of mRNA expression of TOR signal pathway and tight junction proteins at an optimal Arg content of 0.2-0.5 mmol/L.

The solute carrier family 7A, member 7 gene encodes the light chain- y⁺L amino acid transporter-1 (y⁺LAT1) of the heterodimeric carrier responsible for cationic amino acid (CAA) transport across the basolateral membranes of epithelial cells in intestine and kidney. Rising attention has been given to y⁺LAT1 involved in CAA metabolic pathways and growth control. The molecular characterization and function analysis of y⁺LAT1 in grass carp (Ctenopharyngodon idellus) is currently unknown. In the present study, full-length cDNA (2,688 bp), which encodes y⁺LAT1 and contains a 5'-untranslated region (319 bp), an open reading frame (1,506 bp) and a 3'-untranslated region (863 bp), has been cloned from grass carp. Amino acid sequence of grass carp y⁺LAT1 contains 11 transmembrane domains and shows 95 %, 80 % and 75 % sequence similarity to zebra fish, amphibian and mammalian y⁺LAT1, respectively. The tissue distribution and expression regulation by fasting of y⁺LAT1 mRNA were analyzed using real-time PCR. Our results showed that y⁺LAT1 mRNA was highly expressed in midgut, foregut and spleen while weakly expressed in hindgut, kidney, gill, brain, heart, liver and muscle. Nutritional status significantly influenced y⁺LAT1 mRNA expression in fish tissues, such as down-regulation of y⁺LAT1 mRNA expression after fasting (14 days).