Weidan Jiang

In aquaculture, hypoxia is a serious hazard that is frequently reduced by vitamin supplementation. Vitamin A (VA), which fish cannot produce on their own, is vital for their health, but the ability of VA to help fish fight hypoxic stress has not been reported. The study examined the impacts of VA on growth performance, hypoxia-induced liver injury, mitochondrial stress, and endoplasmic reticulum stress in adult grass carp (Ctenopharyngodon idella). Six hundred grass carp (726 ± 1.2 g; 441 d age) were divided into six groups with 4 replicates per group and 25 fish per replicate and fed six diets containing VA levels of 375, 862, 1614, 2099, 2786, and 3118 IU/kg for 60 d to determine growth performance, followed by a 96-h acute hypoxic stress experiment. The results showed that VA (1614-2786 IU/kg) increased final body weight (FBW), percentage weight gain (PWG), and feed efficiency (FE) (P < 0.05). Vitamin A (862-2786 IU/kg) also attenuated the levels of blood parameters (aspartate aminotransferase [AST], alanine aminotransferase [ALT], and cortisol [Cor]) (P < 0.05) and the excessive accumulation of reactive oxygen species (ROS) (P < 0.001) induced by hypoxia. Furthermore, VA (2099-2786 IU/kg) increased the mRNA relative expression levels of mitochondrial biogenesis genes (SDHA, TFAM, PGC1α/β, and NRF1/2) (P < 0.05), while 1614 to 2099 IU/kg of VA decreased the mRNA relative expression levels of mitochondrial stress genes (LONP1, ClpP, Tom40, and FGF21) (P < 0.05), and VA at 1614 IU/kg decreased endoplasmic reticulum stress genes (GRP78, IRE1, XBP1, ATF6, PERK, eIF2α, and CHOP) (P < 0.05). Vitamin A (1614-2786 IU/kg) also enhanced the protein levels of SDHA (P = 0.045) and HSP60/70 (P = 0.001), and VA at 1614 IU/kg reduced the protein levels of FGF21 (P < 0.001), p-PERK (P = 0.002), and p-IRE1 (P < 0.001). In short, intermediate VA supplementation improved growth and alleviated hypoxia-induced mitochondrial and endoplasmic reticulum stress. Moreover, the optimal dietary VA levels for adult grass carp were determined to be 1921 IU/kg for growth (based on weight gain) and 2450 IU/kg for hypoxic stress resistance (based on AST).

Background: Rosemary-derived triterpene acids (TAs), primarily composed of ursolic acid, oleanolic acid, and betulinic acid, exhibit multiple bioactive properties. However, their effects on lipid metabolism and the underlying regulatory pathways remain unclear. This study investigated the effects of dietary supplementation with TAs on the growth performance, digestive and absorptive function, and hepatic lipid metabolism in juvenile grass carp ( Ctenopharyngodon idella ). Methods: In this trial, 2,160 juvenile grass carp (average weight 13.04 ± 0.02 g) were randomly allocated to six dietary treatments, each comprising six replicates with 60 fish per replicate. Fish were fed diets supplemented with increasing concentrations of TAs (0, 58.80, 179.30, 261.90, 312.00, and 390.00 mg/kg) for 70 d. At the end of the trial, relevant samples were collected for subsequent analyses. Results: The results demonstrated that dietary supplementation with TAs significantly increased specific growth rate (SGR), whole-body crude protein (CP) levels, and protein retention value (PRV) in juvenile grass carp, while reducing whole-body ether extract (EE) levels. Moreover, dietary supplementation with TAs significantly enhanced the activities of intestinal digestive enzymes and brush-border enzymes, thereby improving the digestive and absorptive capacity of juvenile grass carp. In the liver, dietary supplementation with TAs markedly inhibited lipid synthesis while promoting lipid utilization. The effects of TAs on lipid metabolism were associated with activation of the hepatic farnesoid X receptor (FXR) pathway, involving peroxisome proliferator-activated receptor alpha (PPARα) and sterol regulatory element-binding protein 1 (SREBP-1). Furthermore, TAs modulated the gut–liver axis by inhibiting the intestinal FXR–sphingomyelin phosphodiesterase 3 (SMPD3)–ceramide pathway, which may contribute to reduced hepatic lipid deposition. Quadratic regression analysis showed that the optimal dietary TAs supplementation levels were 245.00 mg/kg (SGR), 218.33 mg/kg (intestinal lipase activity), and 267.64 mg/kg (hepatic hormone-sensitive lipase activity). Conclusions: The addition of TAs to the diet improved growth performance, digestive and absorptive capacity, and liver lipid utilization in juvenile grass carp. This work reveals the potential application of TAs in aquaculture and provides a theoretical basis for the development of functional feed additives.

Background: Hypoxia is a pervasive challenge in aquaculture that poses a significant threat to aquatic organisms. Since fish cannot synthesize vitamin A endogenously, it must be supplied through diet, and it plays a vital role in supporting fish stress resistance. This study aimed to investigate the protective effects of VA on the gills of adult grass carp ( Ctenopharyngodon idella ) against hypoxia and to elucidate the underlying mechanisms. Methods: Six experimental diets with graded VA levels (375, 862, 1,614, 2,099, 2,786, and 3,118 IU/kg) were fed to grass carp (initial weight: 726 ± 1.2 g) for 60 d. After the trial, 24 fish per treatment were selected, divided equally into normoxic and hypoxic groups, fasted for 24 h, and then subjected to a 96-h acute hypoxic challenge. Results: The results demonstrated that VA supplementation mitigated hypoxia-induced damage in gill tissue, as evidenced by histological examination. Furthermore, VA alleviated oxidative stress, as indicated by reduced levels of lactate (LD), lactate dehydrogenase (LDH), reactive oxygen species (ROS), protein carbonyl (PC), and malondialdehyde (MDA). Further investigations indicated that VA alleviated mitochondrial stress, potentially through suppressing the canonical UPR ^mt axis while activating both the UPR ^mt sirtuin axis and the UPR ^IMS /Erα axis. VA also modulated mitochondrial mass via multiple mechanisms, including the promotion of mitochondrial biogenesis, maintenance of dynamics by stimulating fusion and reducing fission, and inhibition of mitophagy. The suppression of mitophagy likely involved downregulating both the Pink1/Parkin-dependent pathway and the Hif1a-Bnip3 pathway. Taken together, these adaptations suggested an essential role for VA in preserving mitochondrial homeostasis. Based on the quadratic regression analysis of ROS and MDA levels from the hypoxic group, the estimated VA requirements for adult grass carp were 2,013 and 2,056 IU/kg diet, respectively. Conclusions: In summary, this study provided the first evidence that VA conferred protective effects against hypoxia-induced gill damage in grass carp. Graphical

Background: Environmental hypoxia is a common phenomenon in aquaculture, which causes gill tissue injury in fish. Glutathione (GSH) is a vital antioxidant in animal tissues, and its levels decrease under hypoxic conditions. However, the effects of glutathione on fish under hypoxic stress remain poorly understood. This study aimed to investigate the impact of glutathione on gill tissue damage in fish under hypoxic stress and explore the underlying mechanisms. Methods: Six experimental diets with varying glutathione concentrations. The actual glutathione levels in these diets, measured by high-performance liquid chromatography, were 0.00, 145.95, 291.90, 437.85, 583.80, and 729.75 mg/kg, respectively. Fish were fed these diets for 70 d, after which a 96-h hypoxic stress experiment was conducted. The experiment was set up with normoxic and hypoxic groups, in which the dissolved oxygen in the group was 6 mg/L, and that in the group was 1 mg/L. Results: This research revealed that glutathione could enhance the growth performance and antioxidant capability of juvenile grass carp while mitigating the structural damage to gill tissues induced by hypoxia stress. Mechanistic investigations further indicated that glutathione mitigated hypoxia-induced oxidative injury in gill tissues and improved their antioxidant capacity. In addition, glutathione attenuated gill apoptosis induced by hypoxia stress. Glutathione also inhibited the initiation, nucleation, elongation, and degradation phases of autophagy, thereby attenuating hypoxia-induced gill autophagy. Moreover, glutathione was found to alleviate hypoxia-induced endoplasmic reticulum stress (ERS) in gills, a response potentially linked to the suppression of PERK, IRE1, and ATF6 signaling pathways. Finally, based on the ROS and PC contents in gill tissue, the optimum glutathione supplementation levels for juvenile grass carp under hypoxia stress were 437.10 and 495.00 mg/kg, respectively. Conclusions: In conclusion, our experimental results demonstrated the effectiveness of glutathione in alleviating gill tissue damage caused by hypoxic stress. This study confirms the feasibility and effectiveness of dietary glutathione addition to alleviate hypoxic stress in fish.

Skeletal muscle constitutes the largest tissue in fish and represents the primary edible portion. Given the substantial differences in muscle growth dynamics between fish and mammals, fish serve as a crucial and distinctive model organism for investigating the fundamental mechanisms of growth regulation in vertebrates. Myo-inositol (MI), originally discovered in muscle, plays significant biological roles in growth regulation, membrane biogenesis, and transmembrane signal transduction. However, to date, no studies have investigated how MI affects adult fish growth and muscle development. A total of 450 adult grass carp (Ctenopharyngodon idella) (704.84 ± 0.91 g) were randomly divided into 6 treatments with 3 replicates of 25 fish each to receive dietary MI at 35.38 (basal diet, deficient group), 98.12, 195.21, 292.30, 389.39, and 486.48 mg/kg. The trial period lasted for 8 weeks. Results indicated that compared with the 35.38 mg/kg MI group, all groups supplemented with MI improved the specific growth rate (SGR) and percent weight gain (PWG) of adult grass carp (P < 0.05). Compared with the deficient group, the sodium-myo-inositol cotransporter 2 (SMIT2) and MI content in the muscle of grass carp in the 292.30 to 486 mg/kg MI group were significantly elevated (P < 0.05). Compared with the deficient group, the dietary MI levels of 195.21 to 486.48 mg/kg lead to increased myofiber mean diameters and the frequency of myofibers with a diameter > 100 μm, while decreased myofiber diameters < 60 μm (P < 0.05). This implies that MI promotes muscle hypertrophy. The hypertrophic effect of MI was primarily ascribed to an increase in the number of myonuclear and enhanced protein synthesis, which is associated with the regulation of the skeletal muscle lysyl oxidase (LOX) and the protein kinase B (AKT)/target of rapamycin (TOR)/ribosomal protein S6 kinase 1 (S6K1) signaling pathways. Additionally, MI inhibited the myostatin (MSTN) and the forkhead box O3 (FoxO3)/muscle RING-finger protein-1 (MuRF1)/muscle atrophy F-box (MAFbx) pathways, which are involved in muscle atrophy and protein degradation. Based on PWG, the appropriate MI requirement of adult grass carp was determined to be 301.30 mg/kg. This study offers a preliminary theoretical foundation for the potential mechanism by which MI promotes muscle hypertrophy in fish and furnishes a reference for the commercial feed formulation of adult grass carp.

D-mannose, essential for protein glycosylation, has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis. In addition to evaluating growth performance, we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine. All 1800 grass carp (16.20 ± 0.01 g) were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose (0.52, 1.75, 3.02, 4.28, 5.50 and 6.78 g/kg diet) for 70 d. The study revealed that D-mannose increased feed intake (P < 0.001) but did not affect the percent weight gain (PWG), special growth rate, and feed conversion ratio (P > 0.05). D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value (P < 0.05). D-mannose supplementation at 4.28 g/kg increased intestinal length, intestinal weight and fold height of grass carp compared to the control group (P < 0.05). This improvement may be attributed to the phosphomannose isomerase (PMI)-mediated enhancement of glycolysis. This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content (P < 0.05) and improved cellular and intercellular structures for the first time. The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6) signaling pathways. The alleviation of ER stress may be linked to the phosphomannomutase (PMM)-mediated enhancement of protein glycosylation. In addition, ubiquitin-dependent [PTEN-induced putative kinase 1 (PINK1)/Parkin] and ubiquitin-independent [BCL2-interacting protein 3-like (BNIP3L), BCL2-interacting protein 3 (BNIP3), and FUN14 domain containing 1 (FUNDC1)] mitophagy may play a role in maintaining cellular redox homeostasis. The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures, which may be closely associated with the small Rho GTPase protein (RhoA)/the Rho-associated protein kinase (ROCK) signaling pathway. In conclusion, D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways, and enhanced intercellular structures related to tight junctions and adherent junctions. Furthermore, quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg, respectively.