The saving effect of bile acids on cholesterol

Conclusion written at the beginning:
      Adding 0.02% -0.03% bile acids to shrimp low CHO feed can save approximately 0.05% cholesterol. The cholesterol saving effect of bile acids may help reduce shrimp feed costs by 20-30 USD/ton。

Preface:

Cholesterol (CHO) is one of the most important lipids in eukaryotic cells and is crucial for the survival of animals. At the mechanistic level, CHO is responsible for maintaining the strength and integrity of cell membranes, while also participating in the metabolism and transport of lipids in animals, and serving as a precursor framework for steroid hormones, fat soluble vitamins, and bile acids (BA).

In crustaceans, cholesterol accounts for over 90% of total sterols and can regulate crustacean molting and reproduction through the biosynthesis of sesquiterpenes and ecdysteroid hormones. However, crustaceans lack the ability to synthesize cholesterol, so cholesterol is an essential nutrient for crustaceans, mainly obtained through diet< The CHO demand of crustaceans varies depending on their growth stage, feed nutrition, and experimental conditions. Research has found that the optimal dietary CHO level for Macrobrachium rosenbergii is 0.17%, for Penaeus vannamei it is 0.17% to 0.19%, and for juvenile Penaeus vannamei it is 0.16%. In commercial feed, exogenous supplementation of CHO has become a common practice to meet the CHO needs of crustaceans. Adding only 0.2% CHO to the feed will result in an increase of more than 10% in total cost. Overall, CHO has been recognized as one of the most expensive components in crustacean feed, and increasing CHO utilization may help reduce the overall cost of crustaceans.

BA is synthesized by CHO in vertebrates, and the interaction between BA and CHO has been widely reported in vertebrates. In vertebrates, primary BA synthesized by the liver is secreted into bile and ultimately converted into secondary BA by gut microbiota in the small intestine to exert a wider range of biological activities. More than 95% of BA is reabsorbed to initiate the next enterohepatic cycle. Due to its amphiphilic structure, BA can dissolve lipids by forming microcapsules, thereby increasing the utilization of lipids such as cholesterol and fatty acids. In addition, BA can also act as a signaling molecule to regulate the CHO homeostasis in vertebrates by activating nuclear hormone receptors. Previous studies have shown that adding BA to grass carp feed can enhance the protein saving effect of feed fat. Meanwhile, BA may have significant beneficial effects on fish, including improving growth performance and liver function, alleviating stress responses, regulating lipid metabolism and gut microbiota.

For a long time, animal based raw materials such as fish meal, fish oil, and squid oil have been the main source of CHO in traditional feed for crustaceans. In recent years, the use of plant-based materials instead of animal based materials has led to a decrease or insufficient content of CHO in feed. Meanwhile, the use of plant-based ingredients may affect the utilization rate of CHO through anti nutritional factors. In this case, adding emulsifiers to feed to improve the utilization rate of CHO is considered a feasible method to reduce the total cost of aquaculture feed.

Experimental design：
      The aim of this study is to investigate the interaction between CHO and BA in feed on the growth performance, lipid metabolism, immune response, and gut microbiota of Penaeus vannamei, and to explore the saving effect of BA on CHO in shrimp feed. Shrimp with an average initial weight of 1.27 ± 0.18g for 56 consecutive days.

Experimental results and discussion：
      BA is a natural emulsifier that is crucial for the absorption and transport of lipids in vertebrates. In fish research, it has been proven that dietary supplementation with BA can effectively promote the growth performance of salmon, longfin tuna, Nile tuna, and Japanese eel. Unlike fish, crustaceans are unable to synthesize endogenous sterols, so the hydrophobic part of emulsifiers replaces bile acid derivatives with fatty acid chains. Early research has identified several emulsifiers for crustaceans, such as fatty acid acylcarnitine taurine in crabs, fatty acid acyltaurine in river crabs, and fatty acid acyldipeptides in common lobsters. However, the emulsification efficiency of all these endogenous emulsifiers in invertebrates is very low and is considered to be much lower than that of BA. In this study, adding 0.02% -0.03% BA to low CHO feed can save approximately 0.05% CHO. As the CHO level in the feed increases, the CHO saving effect of BA gradually weakens. It is worth noting that shrimp fed with low levels of CHO+BA (feed 5 and feed 6) showed better growth than those fed with high levels of CHO (feed 7) alone. These results indicate that BA can not only promote the digestion of CHO in feed, but also enhance the overall fat utilization efficiency of shrimp. Correspondingly, as an adaptive mechanism for nutrient accumulation in the liver and pancreas, the activity of protease and lipase in BA shrimp significantly increased. The CHO saving effect of BA may help reduce shrimp feed costs by 20-30 US dollars per ton. In summary, our study confirms for the first time the conservation effect of BA on CHO in shrimp feed, which can reduce the feed cost of crustacean aquaculture.
      According to reports, CHO and BA can affect the blood biochemical indicators of crustaceans. TG and TC are important components of blood lipids, which can to some extent reflect the lipid metabolism status of the body. HDL-C and LDL-C play important roles in cholesterol transport in the body. There is a significant positive correlation between serum TC and CHO levels. This study also observed similar results, that is, with the increase of CHO levels, TC and TG showed a significant upward trend. CHO is a biosynthetic precursor of molting hormone in crustaceans, and an increase in TC may contribute to the molting and growth of Penaeus vannamei. In addition, consistent with previous studies on rainbow trout, feed BA significantly reduced TG in shrimp fed with 0.2% and 0.3% CHO, which may be due to the emulsifying effect of BA ① promoting the transport efficiency of TG, ② enhancing the clearance of chylomicrons from the blood or slowing down their release into the blood. In addition, with the increase of HDL-C and the decrease of LDL-C, the increase in BA levels in the diet may help transport cholesterol from tissues to the liver and pancreas. The liver and pancreas are one of the most important organs in crustaceans.
      The liver and pancreas are mainly composed of four types of cells, among which E cells are undifferentiated cells, while B, F, and R cells are responsible for intracellular digestion, synthesis of digestive enzymes, and storage of fat, respectively. In this experiment, the effects of CHO and BA on the structure of the hepatopancreas in crustaceans were explored for the first time. The results indicate that abnormal structural changes in the liver and pancreas were observed in low CHO level feed (feed 1-3), which may be a preliminary response of the liver and pancreas to CHO deficiency. In crustaceans, long-term insufficient intake of CHO not only reduces the stability of cell membrane structure, but also forces the degradation of structural CHO for energy metabolism, ultimately leading to liver and pancreatic cell lysis and necrosis. However, with the increase of BA levels, the liver and pancreas structure of Penaeus vannamei returned to normal.
Cholesterol is considered to play a double-edged sword role in the antioxidant system. By enhancing antioxidant capacity, cholesterol protects cells from oxidative damage. However, excessive CHO may oxidize into CHO oxidation products, leading to lipid metabolism disorders and oxidative stress. In addition, BA can improve the antioxidant status of organisms by directly scavenging hydroxyl radicals and enhancing the activity of antioxidant enzymes. According to reports, adding 0.02-0.03% BA can enhance the antioxidant system of large yellow croaker and largemouth bass. Similarly, in this experiment, the supplementation of BA in the experimental group showed a significant upward trend in T-AOC, SOD, and GSH, indicating that BA can enhance the antioxidant capacity of shrimp, and its effective concentration may be similar to that of fish.
This study indicates that the antibacterial activity in the plasma of shrimp fed with BA feed is enhanced, suggesting that BA may mainly improve shrimp immune response by enhancing humoral immunity, rather than directly affecting total count or phagocytic activity of blood cells. In the intestine, BA stimulates the expression of AMPs through the NF-KB mediated signaling pathway, which may help establish a mucosal barrier and prevent pathogens from invading the intestinal epithelium. In addition, the interaction between BA and CHO in intestinal immune response was observed, and the effective concentration of BA was 0.03% at low CHO levels (0.15% and 0.2%) and 0.02% at high CHO levels (0.3%). We hypothesize that the existence of this interaction may be related to the gut physiological state of different levels of dietary CHO.
Conclusion

In summary, the optimal CHO level for Penaeus vannamei was further determined to be 0.2%, and both CHO and BA in the feed have a promoting effect on the growth performance and fat metabolism of the shrimp. In addition, BA has a CHO conservation effect in shrimp feed and improves shrimp immune response through NF-KB mediated signaling pathway.

Author：Su, Chen, et al.
The original：Interactive effects of dietary cholesterol and bile acids on the growth, lipid metabolism, immune response and intestinal microbiota of Litopenaeus vannamei: Sparing effect of bile acids on cholesterol in shrimp diets
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