Identification, characterization, and expression analysis of adiponectin receptors in black carp Mylopharyngodon piceus in response to dietary carbohydrate.

In the present study, three cDNAs of AdipoRs (MpAdipoR1a, MpAdipoR1b, and MpAdipoR2) were identified from juvenile black carp Mylopharyngodon piceus. There were 375, 378, and 356 amino acids in the MpAdipoR1a, MpAdipoR1b, and MpAdipoR2, respectively. BLAST analysis reveals that MpAdipoRs share high identities with other known AdipoRs from zebrafish, rainbow trout, human, etc. And there were all seven transmembrane regions in the amino acid sequences of MpAdipoR1s and MpAdipoR2, respectively. The relative expression levels of MpAdipoR1s were higher in the liver, blood, brain, and eyes in black carp (p < 0.05). Relatively higher expression of MpAdipoR2 was detected in the liver (p < 0.05) and then in the adipose tissues and blood by real-time PCR assays. The relative expression levels of AdipoR mRNA in the liver, muscle, brain, blood, and adipose tissues were detected by real-time PCR in black carp fed with four levels of dietary carbohydrate (CHO) (10.65, 19.43, 28.84, and 37.91%) for 9 weeks, respectively. The expression levels of MpAdipoR1s in the liver, muscle, brain, and blood were induced and reached to the maximum at optimal dietary CHO (19.43 or 28.84%) and then were decreased at 37.91% dietary CHO, although there were no significant differences on the expression levels of MpAdipoR1a in the liver between 19.43, 28.84, and 37.91% dietary CHO. Similarly, MpAdipoR2 were also induced to the maximal levels in the liver, muscle, brain, and blood at optimal dietary CHO (19.43 or 28.84%) compared with that at 10.65% dietary CHO, although no significant differences were observed on the expression levels of MpAdipoR2 in the liver and muscle between groups fed 19.43 and 37.91% dietary CHO (p > 0.05). However, the expression levels of MpAdipoRs in the adipose tissues were significantly downregulated at excessive dietary CHO (37.91%) compared with other groups (p < 0.05). These results indicated that MpAdipoRs were inducible proteins and might enhance the understanding of their vital roles in the regulation of glucose metabolic homeostasis in black carp.

The effects of dietary leucine on the growth performances, body composition, metabolic abilities and innate immune responses in black carp Mylopharyngodon piceus.

The present study was focused on the growth, body composition, metabolic abilities and innate immune responses in juvenile black carp Mylopharyngodon piceus fed with six levels of dietary leucine (7.3, 12.4, 16.2, 21.9, 28.3 and 34.5 g kg-1) for 9 weeks. Results showed that the highest weight gain (WG) and the lowest feed conversion ratio (FCR) was obtained at 23.5 and 23.9 g kg-1 dietary leucine using second-order polynomial model, respectively. Adequate dietary leucine content (21.9 and 28.3 g kg-1) could significantly up-regulate the expression levels of neuropeptide Y (NPY) and ghrelin (GRL) in the brain of black carp juveniles. The protein efficiency ratio (PER), feed efficiency ratio (FER) and protein deposition ratio (PDR) were also significantly increased by adequate dietary leucine content (21.9 and 28.3 g kg-1) (p < 0.05). Adequate dietary leucine content (21.9 and 28.3 g kg-1) could significantly up-regulate the activities of metabolic enzymes, such as α amylase, trypsin, chymotrypsin and elastase in the liver of Black carp (p < 0.05). However, the activities of alanine transaminase (ALT), aspartate aminotransferase (AST) and leucine aminopeptidase (LAP) were significantly reduced in the fish serum by adequate dietary leucine content (21.9 and 28.3 g kg-1) compared with leucine-deficient diet (7.3 and 12.4 g kg-1). In addition, 21.9 and 28.3 g kg-1 dietary leucine could significantly increase complement component 3 (C3) and C4 contents, lysozyme (LYZ) activities in the serum compared with the leucine-deficient diet (7.3 and 12.4 g kg-1) (p < 0.05). Furthermore, optimal dietary leucine could also significantly up-regulate the mRNA expression levels of LYZ, interferon α (IFN-α), hepcidin (HEPC), natural resistance-associated macrophage protein (NRAMP), C3 and C9 in the blood of juvenile black carp compared with the leucine-deficient diets (7.3 and 12.4 g kg-1) (p < 0.05). In conclusion, these results suggest that adequate dietary leucine (21.9 and 28.3 g kg-1) could increase growth performances, improve metabolic abilities and then enhance non-specific immunities in black carp juveniles.

Molecular cloning, characterization and mRNA expression of six peroxiredoxins from Black carp Mylopharyngodon piceus in response to lipopolysaccharide challenge or dietary carbohydrate.

Peroxiredoxin (Prx) belongs to a cellular antioxidant protein family that plays important roles in innate immune function and anti-oxidative capability. In the present study, six Prxs were cloned from Black carp Mylopharyngodon piceus (MpPrx) by homology cloning and rapid amplification of cDNA ends (RACE) techniques. There were 199, 197, 250, 260, 189 and 222 amino acids in six MpPrxs, respectively. BLAST analysis reveals that MpPrxs shares high identities and similar characteristics with other known Prxs from animals. The phylogenetic analysis evidenced three major subclasses corresponding to one-Cys-Prx (MpPrx6), typical two-Cys-Prx (MpPrx1-4) and atypical 2-Cys-Prx (MpPrx5) that reflected the present hierarchy of vertebrates and invertebrates. Although six MpPrxs are constitutively expressed in all tissues, relatively higher-level mRNA expression levels of six MpPrxs can be detected in liver, eyes, heart and adipose tissues by real-time PCR assays. The transcriptional patterns of six MpPrxs mRNA in liver were detected by real-time PCR in Black carp after lipopolysaccharide (LPS) challenge and treated with graded levels of dietary carbohydrate (CHO) (106.5, 194.3, 288.4 and 379.1 g kg(-1)), respectively. These results showed that stimulation with LPS could induce up-expression of six MpPrxs mRNA, and the variations of MpPrx4 were more sensitive than these of other MpPrxs in the liver of Black carp. Compared with those in group with 106.5 g kg(-1) dietary CHO, the expression levels of MpPrx2, MpPrx3 and MpPrx6 were significantly down-regulated while MpPrx5 were significantly induced in liver of Black carp fed with adequate dietary CHO (194.3 g kg(-1)). In addition, significant up-regulations of MpPrx2, MpPrx3 and MpPrx6 were observed in Black carp fed with excessive dietary CHO (379.1 g kg(-1)). And MpPrx4 could be constantly induced with increasing dietary CHO contents in this study. These results indicated that MpPrxs were constitutive and inducible proteins and might play important roles in innate immune function after LPS challenge and regulating redox homeostasis in the metabolism of dietary CHO.

The effects of dietary carbohydrate on the growth, antioxidant capacities, innate immune responses and pathogen resistance of juvenile Black carp Mylopharyngodon piceus.

The present study was focused on the growth, antioxidant capacities, innate immune responses and pathogen resistance in juvenile Black carp Mylopharyngodon piceus fed with graded levels of dietary carbohydrate (CHO) (0.6, 106.5, 194.3, 288.4, 379.1 and 473.8 g kg(-1)) for 9 weeks. Results showed that highest weight gain and special growth ratio was obtained at 288.4 g kg(-1) dietary CHO. And adequate dietary CHO content (288.4 g kg(-1)) could significantly increase the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx), promote reduced glutathione (GSH) content and then increase the total antioxidant capacities (TAOC) in the liver of M. piceus. However, the malondialdehyde (MDA) levels in the fish liver could be significantly aggravated by excessive dietary CHO. Serum cortisol (COL) levels could be significantly increased in juvenile Black carp M. piceus fed with 379.1 g kg(-1) dietary CHO compared with CHO-deficient diets. Activities of alanine transaminase (GPT) and aspartate transaminase (GOT) were both decreased in the serum of juvenile Black carp M. piceus fed with 194.3 g kg(-1) dietary CHO compared with CHO-deficient diets (0.6 and 106.5 g kg(-1)) or CHO-excess diets (379.1 and 473.8 g kg(-1)). In addition, 288.4 g kg(-1) dietary CHO could significantly up-regulate the mRNA expression levels of hepcidin (HEPC), natural resistance-associated macrophage protein (NRAMP), tumor necrosis factor-α (TNF-α) and interferon (IFN), lysozyme (LYZ) and complement component 3 (C3) in the blood and liver samples of juvenile Black carp M. piceus compared with the CHO-deficient diets (0.6 and 106.5 g kg(-1)). Moreover, 288.4 g kg(-1) dietary CHO could also enhance the contents of C3 and plasma nitrogen monoxide (NO), and increase the activities of LYZ and total nitric oxide synthase (t-NOS) in the serum compared with the CHO-deficient or CHO-excess diets. Furthermore, the survival rates were also increased by adequate dietary CHO (194.3 and 288.4 g kg(-1)) fed to juvenile Black carp M. piceus after infection with Aeromonas hydrophila. In conclusion, these results suggest that adequate dietary CHO (288.4 g kg(-1)) could increase growth, reduce oxidative stress, enhance innate immune responses, improve the health states and then promote disease resistance in juvenile Black carp M. piceus.