WO2020011128A1 - Bile acid, preparation method thereof, and application of same in preparing animal feed additive - Google Patents

Bile acid, preparation method thereof, and application of same in preparing animal feed additive Download PDF

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WO2020011128A1
WO2020011128A1 PCT/CN2019/095091 CN2019095091W WO2020011128A1 WO 2020011128 A1 WO2020011128 A1 WO 2020011128A1 CN 2019095091 W CN2019095091 W CN 2019095091W WO 2020011128 A1 WO2020011128 A1 WO 2020011128A1
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bile acid
feed
bile
acid
preparation
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PCT/CN2019/095091
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French (fr)
Chinese (zh)
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曹爱智
籍立民
娄倩倩
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山东龙昌动物保健品有限公司
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Priority claimed from CN201810747818.8A external-priority patent/CN108522860A/en
Priority claimed from CN201810747819.2A external-priority patent/CN108783015A/en
Priority claimed from CN201810747805.0A external-priority patent/CN108522858A/en
Application filed by 山东龙昌动物保健品有限公司 filed Critical 山东龙昌动物保健品有限公司
Publication of WO2020011128A1 publication Critical patent/WO2020011128A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds

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  • the invention belongs to the field of animal feed additives, and in particular relates to a bile acid, a preparation method thereof, and an application in preparing animal feed additives.
  • Bullfrog (Rana catesbeiana) has the advantages of fast growth, delicious taste, rich nutrition, high protein content, etc., which is loved by consumers and has a broad market. And because of its high yield, short breeding cycle, good economic benefits, strong adaptability and other advantages, in recent years the bullfrog breeding industry has developed rapidly in various provinces in southeast China. However, under intensive breeding conditions, due to a large amount of compound feed and lack of exercise, bullfrogs generally have high body fat, which not only reduces the quality and edible proportion of bullfrogs, increases the cost of breeding, but also causes diseases. Therefore, adding a lipid-lowering factor to feed to reduce body fat deposition is becoming a hot research topic in aquatic animal nutrition and feed science.
  • the object of the present invention is to provide a bile acid, a preparation method thereof, and an application thereof for preparing an animal feed additive.
  • the bile acid according to the present invention is added to an animal's diet, and the growth performance of the animal is significantly improved. To improve the antioxidant capacity.
  • the invention provides the application of bile acid in the preparation of an additive for improving the growth performance of weaned piglets.
  • the bile acid includes the following components: in terms of mass ratio, 5% to 20% of cholic acid, 55% to 75% of hyodeoxycholic acid, and 10% to 20% of chenodeoxycholic acid.
  • the method for preparing the bile acid includes the following steps:
  • the filtered white solids After filtering the white solids, rinse with water, and the water to be filtered will be neutral when the washing ends; the filtered white solids are dried in an oven to a moisture content of not more than 10%, and a crude bile acid is obtained;
  • the obtained bile acid is put into an oven, and is subjected to vacuum drying through steam.
  • the bile acid moisture does not exceed 1%, the bile acid is obtained.
  • weaned piglets are piglets 1-2 weeks after weaning.
  • the bile acid is added to a diet containing rice bran oil and / or full fat soybean meal.
  • the added amount of the bile acid is 0.03% to 0.1% of the total mass of the diet.
  • the mass ratio of the amount of rice bran oil to the total diet is 0.7%
  • the daily weight gain of weaned piglets is the highest
  • the feed-to-meat ratio is the lowest.
  • the invention also provides the application of bile acid in the preparation of feed additive for bullfrog.
  • the invention also provides the application of bile acid in the preparation of feed additives for largemouth bass.
  • the added amount of bile acid in the largemouth bass feed is 200mg / kg-450mg / kg.
  • the present invention proves through experiments that adding bile acid to the diet of weaned piglets can improve its growth performance, improve the digestibility of nutrients, especially fat, and reduce the plant Anti-nutritional factors of protein, thus comprehensively improve the growth performance of piglets, improve feed utilization and uniformity of final weight, reduce meat production costs, and improve economic returns.
  • the invention proves through experiments that the addition of bile acid to the feed can increase the intestinal protease and lipase activities of the bullfrog, promote the digestion and absorption of feed nutrients, and increase the apparent digestibility of the bullfrog to dry matter, protein and fat in the feed, thereby improving Weight gain and feed efficiency.
  • Adding bile acid to the feed can reduce the catabolism of bullfrog body protein, promote protein deposition and lipolysis, thereby reducing the ratio of visceral body, increasing the edible proportion of bullfrog, and increasing the body protein content.
  • Adding bile acid to the feed can reduce the fat level and malondialdehyde content of the bullfrog liver, keep the liver at a low oxidative stress level, thereby maintaining healthy liver tissue structure and saving antioxidant energy expenditure.
  • the experiment proves that adding appropriate amount of bile acid to the feed can effectively promote feeding and growth performance.
  • the analysis of variance shows that adding 300 mg / kg bile acid to the feed, large mouth bass has the highest growth performance, and the liver bile acid content is not the same as that of the D240 group. difference.
  • bile acid effectively reduces the MDA content in the liver of largemouth bass and improves the antioxidant capacity; by increasing the HDL-C content in the plasma, it significantly promotes fat metabolism and effectively improves the intestinal amylase and lipase activities.
  • the invention proves the feasibility of adding bile acid to the largemouth bass feed through experiments, and has a good market application prospect.
  • Figure 1 is a section of bullfrog liver tissue treated with 100 mg / kg bile acid added to the feed.
  • Figure 2 is a section of bullfrog liver tissue treated with 200 mg / kg bile acid added to the feed.
  • Figure 3 is a section of bullfrog liver tissue treated with 300 mg / kg bile acid added to the feed.
  • Figure 4 is a section of bullfrog liver tissue treated with 400 mg / kg bile acid added to the feed.
  • Figure 5 shows the effect of bile acid content on the specific growth rate of largemouth bass
  • Figure 6 shows the effect of bile acid content on the feeding rate of largemouth bass
  • Figure 7 shows the effect of bile acid content in feed on total bile acid content in the liver of largemouth bass.
  • the present invention performs the following tests.
  • the purpose of the test of the present invention is to evaluate the effect of adding bile acids to diets containing rice bran oil, whole fat soybean meal, and a mixture of the two on the growth performance of piglets.
  • the bile acid according to the present invention is prepared through five steps of saponification, decolorization, acidification, purification and recrystallization. The detailed steps are as follows:
  • the solution After being driven into the acidification tank through a pipeline centrifugal pump, the solution was cooled to room temperature by passing condensed water through the interlayer of the tank body. Slowly add a 5% hydrochloric acid solution to the acidification tank through the acid metering tank and keep stirring. When the pH of the solution is around 3, acid addition is stopped, and a white solid is obtained at this time.
  • the white solid was filtered with a plate and frame filter. After filtering, rinse with water. The water to be filtered out is neutral and the washing is finished. The purpose of water washing is to remove water-soluble impurities attached to the surface of bile acid solids.
  • the filtered white solid was placed in an oven and dried at 100 ° C to a moisture content of no more than 10% to obtain a crude bile acid.
  • the obtained bile acid was tiled in a stainless steel drying dish, put into an oven, and vacuum-dried with steam, and the drying temperature was 100 ° C.
  • the bile acid turns into a white powder, sampling and testing, if the moisture does not exceed 1%, it is considered to be dry.
  • the trial was conducted at a well-regulated commercial pig farm (Reang Sifi Farm) in Khon Kaenzhou, Thailand. 480 28-day-old weaned three-way crossbred (Dulock ⁇ about g ⁇ Changbai) piglets (average weight 8.62–8.65 kg) were divided into 4 treatments, each treatment was repeated 4 times, each 30 replicates, male and female half.
  • the piglets were housed in a nursery house with spray-adjustable temperature, and the test period was 28-62 days old, with free feeding and drinking water. Each house has 3 10-hole feeders and 4 nipple drinkers. Weigh the start and end weight of the test piglets, and record the feed intake and mortality daily until the end of the test.
  • Piglet uniformity is a percentage based on 2 times the standard deviation of the mean final weight.
  • Test group 1 contained rice bran oil and full-fat soybeans, and no bile acid was added.
  • the other three test groups were supplemented with 0.04% bile acid (the mass ratio of bile acid to the total feed was 0.04%), and the mass ratio of rice bran oil to the total feed was 0.7%, 1.0% and 1.3%, respectively, and crushed rice Supplement (see Table 1 for details).
  • the diet was fed in a paste form.
  • test results of the effects of bile acids on the growth performance of piglets are shown in Table 2.
  • the diet of the test group was supplemented with 0.04% bile acid
  • the amount of rice bran oil was 0.70%, 1.00% and 1.3%
  • the test period was 34 days.
  • the addition of 0.04% bile acid to the piglet diet can significantly improve growth performance, significantly reduce FCR (P ⁇ 0.05); increase FI (P> 0.05); improve UBWT (P> 0.05); significantly reduce FCG ( P ⁇ 0.05).
  • Addition of bile acids to the piglet diet can increase net profit, of which the addition of 0.7% rice bran oil has the highest return on investment (ROI).
  • adding 0.04% bile acid and 0.7% rice bran oil to the piglet diet can improve piglet growth performance, increase BWG, SR and UBWT, reduce FCR and FCG, and increase ROI.
  • the three test groups of rice bran oil dosage (0.70%, 1.00%, 1.30%), adding 0.04% bile acid can increase body weight and weight gain, but the difference between different rice bran oil dosage groups is not the same Significant (P> 0.05).
  • P> 0.05 Remarkably reduce the ratio of feed to meat, increase the survival rate, and improve the uniformity of the final weight, but the difference between the different rice bran oil dosage groups is not significant, which significantly reduces the cost of meat production.
  • the experimental results of the present invention show that adding bile acid can optimize the digestion and absorption of feed for piglets, in which the experimental group that reduced 0.7% rice bran oil has the highest daily weight gain, the lowest feed-to-meat ratio, and the highest economic benefit return.
  • the results also showed that the return of economic benefits of the three experimental groups added with bile acid was higher than that of the control group, and the experimental group that reduced 0.7% rice bran oil had the highest return on investment.
  • Weaned present invention used per kg diet contains: vitamin A: 2500IU .; Vitamin D 3: 250IU; Vitamin E: 20IU; Vitamin B 12: 0.2mg; pantothenic acid: 12 mg of; Niacin: 20mg; riboflavin Su: 4mg; thiamine: 2mg; choline chloride 1g; vitamin B 6: 2mg; biotin: 0.3mg; vitamin K 3: 0.5mg; folic acid: 0.3mg; ethoxyquin: 12.5mg; copper : 250mg; Iodine: 0.4mg; Iron: 100mg; Manganese: 40mg; Selenium: 0.3mg; Zinc: 100mg; Cobalt: 0.14mg; : 200 mg; chlortetracycline: 400 mg.
  • Average net profit per head weight gain ⁇ unit price (82 baht / kg)-feed cost (baht / head)
  • the test results of the present invention show that compared with the control group without added bile acid, the growth performance of the weaned piglets during the whole period of the experimental period (34d) can be improved by adding bile acid, and the test group added 0.7% rice bran oil daily.
  • the present invention adds bile acid to the diet of weaned piglets, which can improve the digestibility of nutrients, especially fat, and reduce the anti-nutritional factors of plant proteins, thereby comprehensively improving the growth performance of piglets, increasing the utilization rate of feed and the uniformity of final weight, reducing the production Meat costs increase economic returns.
  • the experimental frogs were purchased from the same batch of bullfrogs cultivated in a farm in Tongan District, Xiamen. Before the formal experiment, the bullfrog was kept in an indoor holding tank and fed with experimental control feed to make it gradually adapt to the experimental feed and breeding environment. After 15 days of temporary breeding, the bullfrogs were starved for 24 hours, and healthy bullfrogs of uniform size were randomly allocated to 12 aquariums (150L) in the indoor breeding system for breeding experiments.
  • the initial weight of the experimental bullfrog was (75 ⁇ 4.23) g, which was randomly divided into 4 groups, each group had 3 repetitions, and each restocked 12 bullfrogs. Farming experiments lasted 8 weeks. According to the feeding situation of bullfrogs in each tank, adjust to achieve full feeding. Feed 2 times a day (8:00, 18:00), and change the water and clean the tanks 30 minutes before feeding. Feeding and death of bullfrogs were recorded during breeding.
  • Bouin's fixative was used to fix the bullfrog liver, and the sections were sliced by conventional paraffin sectioning with a thickness of 7-9 ⁇ m, and stained with HE. Observe under a Nikon eslipse 50i microscope and take pictures with a Nikon Coolpix 5400 digital camera.
  • the bullfrog overall, muscle and liver components are shown in Table 5. There was no significant difference in the water and crude ash content of the bullfrogs in each group (P> 0.05), but the crude fat content of the bullfrogs in the bile acid addition group was significantly lower than that in the control group.
  • the bile acid addition amount was 100, 200, and 300 mg / kg, Compared with the control group, the total crude fat content was reduced by 12.69%, 13.46%, and 15.38% (P ⁇ 0.05).
  • the increase of the amount of bile acid the content of crude protein increased compared with the control group.
  • the amount of bile acid was 200 mg At kg / kg, the total crude protein content was significantly higher than the control group (P ⁇ 0.05).
  • Adding 200mg / kg bile acid to the feed can significantly increase the apparent digestibility of dry matter, protein and fat of the bullfrog (P ⁇ 0.05), while the digestibility of the other bile acid addition groups is higher than the control group, but the difference is Not significant level (P> 0.05).
  • the experimental results are shown in Figures 1-4. According to the experimental results, compared with the control group, when bile acids were added to the feed at 100 mg / kg, 200 mg / kg, 300 mg / kg, and 400 mg / kg, the liver cells were arranged neatly. The intercellular space is uniform.
  • Adding bile acid to the feed can reduce the catabolism of bullfrog body protein, promote protein deposition and lipolysis, thereby reducing the ratio of visceral body, increasing the edible proportion of bullfrog, and increasing the body protein content.
  • Adding bile acid to the feed can reduce the fat level and malondialdehyde content of the bullfrog liver, keep the liver at a low oxidative stress level, thereby maintaining healthy liver tissue structure and saving antioxidant energy expenditure.
  • the target animal used in the test was the largemouth bass (Micropterussalmoides), which was purchased from Foshan Sanshui Platinum Aquatic Seeds Co., Ltd. Before the test officially started, the test fish were temporarily kept in the breeding system for 2 weeks. Source of source bile acids.
  • Treatment and preventive measures (cause, time, type, duration, etc.): 2% saline solution was used to disinfect the experimental fish and the breeding system before the experiment. No disease occurred during the trial.
  • the bile acids used in the test include pig bile acid, pig deoxycholic acid, and chenodeoxycholic acid.
  • the bile acids described in the present invention are prepared through five steps: saponification, decolorization, acidification, purification, and recrystallization. The detailed steps are as in the examples. 1.
  • Bile acids of 0, 80, 160, 240, 300, and 600 mg / kg were added to the basic feed of largemouth bass, respectively, and 6 kinds of extruded and extruded floating pellets with a diameter of 2.0 mm were prepared, and then dried naturally for later use.
  • the six processing groups are named D0, D80, D160, D240, D300, and D600 in this order.
  • the trial was conducted in an indoor circulating flow aquaculture system of the National Aquatic Feed Safety Evaluation Base (Beijing). Largemouth bass with a healthy body and an even individual (starting average body weight 6.17 ⁇ 0.02g) was randomly selected and allocated to a conical breeding barrel with a volume of 0.26m. This experiment was designed with 5 treatment groups, each with 6 repetitions and 30 fish per barrel.
  • the breeding trials lasted for 70 days from July 14, 2014 to September 21, 2014.
  • Relative weight gain rate 100 ⁇ (average weight of the last fish-average weight of the initial fish + weight of the dead fish) / average weight of the initial fish;
  • Feed coefficient (FCR) food intake / (last fish weight + dead fish weight-initial fish weight);
  • Three fish were randomly selected from each barrel to measure body length, body weight, internal organ weight, liver weight, and spleen weight.
  • Hepatosomal ratio 100 ⁇ liver weight / whole fish weight
  • Viscerasomatic index 100 ⁇ visceral weight / body weight
  • Spleen index 100 ⁇ spleen weight / whole fish weight
  • Detection indicators are: plasma lipid metabolism indicators: total cholesterol (TC), triglycerides (TG), total bilirubin (TBILI), high density lipoprotein (IHDL), alkaline phosphatase (ALP); plasma antioxidant indicators : Malondialdehyde (MDA), total superoxide dismutase (SOD), total antioxidant capacity (T-AOC), glutathione-S transferase per (GST), glutathione peroxidase ( GSHI-PX) and so on.
  • TC total cholesterol
  • TG total triglycerides
  • TILI total bilirubin
  • IHDL high density lipoprotein
  • ALP alkaline phosphatase
  • plasma antioxidant indicators Malondialdehyde (MDA), total superoxide dismutase (SOD), total antioxidant capacity (T-AOC), glutathione-S transferase per (GST), glutathione peroxidase ( GSHI-PX)
  • livers of 2 fishes were randomly collected from each bucket and stored in a refrigerator at -80 ° C.
  • Preparation of liver tissue samples Take 03-0.5g of tissue, add media to homogenize (the homogenized medium is determined according to the detection index), and after the tissue is fully crushed and mixed, centrifuge at 3000 mp for 10 minutes, and take the supernatant for testing.
  • test data were expressed as mean ⁇ standard error (Mean ⁇ SE). All data were analyzed by one-way analysis of variance (ANOVA) using SPSS Statistics 17.0 software. Duncan's multiple comparison test was used to test the significance of the differences. The significance level was P ⁇ 0.05. meter.
  • the effects of adding bile acids to the feed on the growth performance of largemouth bass are shown in Table 13.
  • the survival rate of largemouth bass was higher than 96.67% in each group, and there was no significant difference between the groups.
  • the end weight of D300 group was significantly higher than that of other groups, and its WGR and SGR also reached the highest, and were significantly higher than those of control group and D80 and D160 groups.
  • the feeding rate of this group also reached the highest level, and was significantly higher than that of the control group (P ⁇ 0.05). There were no significant differences in feed coefficients among the groups (P> -0.05).
  • regression model analysis was performed, and the optimal amounts of bile acids in the feed were 440.5 mg / kg and 283.3 mg / kg ( Figures 5 and 6).
  • the effects of adding bile acid to the feed on body index of largemouth bass are shown in Table 14.
  • the liver-body ratio increased first and then decreased.
  • the liver-body ratio of the D80 group was significantly higher than that of the D240, D300, and D600 groups, and there was no significant difference from the control group. There was no significant difference in fatness, spleen index, and visceral body ratio among the 6 treatment groups (P> 0.05).
  • the midgut amylase content of largemouth bass showed an irregular upward trend, reaching the highest in the D300 group (P ⁇ 0.05), and the midgut lipase showed irregular differences (P ⁇ 0.05).
  • the hindgut amylase reached the highest at D600, which was significantly higher than other groups; the hindgut fat increased first and then decreased, and the D80 group reached the highest, but there was no significant difference between each group and the control group.
  • the gallbladder bile acid content in each group showed a significant upward trend, and the highest gallbladder bile acid content in the D600 group was about 4.9 times that of the control group.
  • the present invention tests the following items through experiments: growth performance: survival rate, specific growth rate, weight gain rate, feed coefficient, feeding rate, fullness, liver-body ratio, viscera-body ratio; plasma fat metabolism and antioxidant performance: ALP , TPRO, ALB, TBILI, TC, TG, HDL, SOD, MDA, T-AOC, GST, GST-PX; and the bile acid content of the liver and gallbladder.
  • the test results of the present invention show that adding a proper amount of bile acid to the feed can effectively promote feeding and growth performance, but at the same time increase the total bile acid content of the liver.
  • the optimal bile acid additions in the feed for largemouth bass were 440.5 mg / kg, 283.3 mg / kg, and 253.8 mg / kg by regression model analysis. .
  • the analysis of variance showed that adding 300mg / kg bile acid to the feed, the maximum growth performance of largemouth bass was obtained, and the content of liver bile acid was not different from that of D240 group.
  • bile acid effectively reduces the MDA content in the liver of largemouth bass and improves the antioxidant capacity; by increasing the HDL-C content in the plasma, it significantly promotes fat metabolism and effectively improves intestinal amylase and lipase activities.
  • Comprehensive growth performance, immune anti-oxidation index and bile acid accumulation level in the liver, the recommended amount of bile acid in largemouth bass feed is 253.8mg / kg-283.3mg / kg, the maximum recommended dose is 300mg / kg.

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Abstract

An application of a bile acid in preparing an animal feed additive. The bile acid comprises hyocholic acid, hyodeoxycholic acid, and chenodeoxycholic acid. The animal feed additive can improve animal growth performance, efficiency of feed utilization, and uniformity of body weight at termination and reduce feeding cost of gain.

Description

一种胆汁酸及其制备方法和在用于制备动物饲料添加剂中的应用Bile acid, preparation method thereof and application in preparation of animal feed additive 技术领域Technical field
本发明属于动物饲料添加剂领域，具体涉及一种胆汁酸及其制备方法和在用于制备动物饲料添加剂中的应用。The invention belongs to the field of animal feed additives, and in particular relates to a bile acid, a preparation method thereof, and an application in preparing animal feed additives.
背景技术Background technique
众所周知，生长早期的仔猪胃肠道发育不完善。研究表明：生长早期的仔猪胃内不能分泌充足的盐酸，胃内pH高，不能激活胃蛋白酶原，导致有害菌侵入肠道上段。如果仔猪突然由母乳更换为谷物日粮，由于肠道消化能力不足，谷物日粮进入大肠发酵。大肠病菌滋生，挥发性脂肪酸和其他电解质聚积，导致渗透失衡，引起仔猪腹泻。因此，当仔猪日粮变化时，必须添加适当的酸化剂以保持正常的消化功能。然而，必须认识到脂肪的吸收率是随着日龄而增长的，仔猪对脂肪的吸收有生理性限制。It is well known that gastrointestinal development of piglets in early growth is incomplete. Studies have shown that piglets in early growth cannot secrete sufficient hydrochloric acid in their stomachs, the stomach pH is high, and pepsinogen cannot be activated, causing harmful bacteria to invade the upper intestine. If a piglet suddenly changes from breast milk to a cereal diet, the cereal diet enters the large intestine for fermentation due to inadequate intestinal digestion. The growth of coliform bacteria, accumulation of volatile fatty acids and other electrolytes, leads to imbalance of penetration and causes diarrhea in piglets. Therefore, when the piglet diet changes, an appropriate acidulant must be added to maintain normal digestive function. However, it must be recognized that fat absorption increases with age, and piglets have physiological limitations in fat absorption.
牛蛙(Rana catesbeiana)具有生长快、味道鲜美、营养丰富、蛋白含量高等优点，深受消费者喜爱，拥有广阔的市场。又因其产量高、养殖周期短、经济效益好、适应性强等优点，近年来牛蛙养殖业在我国东南各省迅猛发展。然而在集约化养殖条件下，由于大量摄食配合饲料，缺少运动，导致牛蛙普遍出现体脂肪偏高，这不仅降低了牛蛙的品质和可食比例，提高了养殖成本，更易造成疾病的发生。因此，在饲料中添加一种降脂因子，降低体脂肪沉积正成为水产动物营养与饲料学的研究热点。Bullfrog (Rana catesbeiana) has the advantages of fast growth, delicious taste, rich nutrition, high protein content, etc., which is loved by consumers and has a broad market. And because of its high yield, short breeding cycle, good economic benefits, strong adaptability and other advantages, in recent years the bullfrog breeding industry has developed rapidly in various provinces in southeast China. However, under intensive breeding conditions, due to a large amount of compound feed and lack of exercise, bullfrogs generally have high body fat, which not only reduces the quality and edible proportion of bullfrogs, increases the cost of breeding, but also causes diseases. Therefore, adding a lipid-lowering factor to feed to reduce body fat deposition is becoming a hot research topic in aquatic animal nutrition and feed science.
发明内容Summary of the invention
本发明的目的是提供了一种胆汁酸及其制备方法和在用于制备动物饲料添加剂中的应用，将本发明所述的胆汁酸添加到动物的日粮中，显著提高了动物的生长性能，提高了抗氧化能力。The object of the present invention is to provide a bile acid, a preparation method thereof, and an application thereof for preparing an animal feed additive. The bile acid according to the present invention is added to an animal's diet, and the growth performance of the animal is significantly improved. To improve the antioxidant capacity.
为实现上述发明目的，本发明采用下述技术方案予以实现：In order to achieve the above-mentioned object of the present invention, the present invention is implemented by using the following technical solutions:
本发明提供了胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用。The invention provides the application of bile acid in the preparation of an additive for improving the growth performance of weaned piglets.
所述胆汁酸包括以下成分：按质量比计，猪胆酸5％-20％、猪去氧胆酸55％-75％和鹅去氧胆酸10％-20％。The bile acid includes the following components: in terms of mass ratio, 5% to 20% of cholic acid, 55% to 75% of hyodeoxycholic acid, and 10% to 20% of chenodeoxycholic acid.
所述胆汁酸的制备方法包括如下步骤：The method for preparing the bile acid includes the following steps:
(1)皂化(1) Saponification
称取粉碎的胆膏，加入反应釜中，加入氢氧化钠溶液，升温并保持沸腾状态6-24h，将皂化液冷却至固液分层，将上清液去除，固体即为胆膏皂化品；Weigh the pulverized bile, add it to the reaction kettle, add sodium hydroxide solution, heat up and keep boiling for 6-24h, cool the saponified liquid to solid-liquid layering, and remove the supernatant. The solid is the saponified bile. ;
(2)脱色(2) Discoloration
向胆膏皂化品中加水并加热使胆膏皂化品完全溶解，然后加入双氧水，搅拌均匀后反应，反应完毕后，将反应溶液过滤得滤液；Add water to the gall saponified product and heat to completely dissolve the gall saponified product, then add hydrogen peroxide, stir and react after the reaction is uniform. After the reaction is completed, filter the reaction solution to obtain a filtrate;
(3)酸化(3) Acidification
将所述滤液冷却至室温，缓慢加入盐酸溶液，并搅拌，待溶液pH值在3-4时，停止加酸，此时得到白色固形物；The filtrate was cooled to room temperature, and the hydrochloric acid solution was slowly added and stirred. When the pH of the solution was 3-4, the addition of acid was stopped, and a white solid was obtained at this time;
(4)纯化(4) Purification
将所述白色固形物过滤后，加水冲洗，待过滤出的水检测呈中性为水洗结束；将过滤后的白色固形物放入烘箱中干燥至水分不高于10％，得胆汁酸粗品；After filtering the white solids, rinse with water, and the water to be filtered will be neutral when the washing ends; the filtered white solids are dried in an oven to a moisture content of not more than 10%, and a crude bile acid is obtained;
(5)重结晶(5) Recrystallization
将得到的胆汁酸粗品投入萃取罐中，加入乙酸乙酯搅拌溶解，待胆汁酸粗品基本溶解，加入无水硫酸钠脱水，收集溶液，将溶液进行减压浓缩，将浓缩液压滤得到胆汁酸；Put the obtained crude bile acid into an extraction tank, add ethyl acetate and stir to dissolve. After the crude bile acid is basically dissolved, add anhydrous sodium sulfate to dehydrate, collect the solution, concentrate the solution under reduced pressure, and concentrate the hydraulic filtration to obtain bile acid;
(6)干燥(6) Drying
将得到的胆汁酸放入烘箱，通蒸汽进行真空干燥，待胆汁酸水分不超过1％，则得到所述胆汁酸。The obtained bile acid is put into an oven, and is subjected to vacuum drying through steam. When the bile acid moisture does not exceed 1%, the bile acid is obtained.
进一步的：所述断奶仔猪为断奶后1-2周的仔猪。Further: the weaned piglets are piglets 1-2 weeks after weaning.
进一步的：所述胆汁酸添加到含有米糠油和/或全脂豆粕的日粮中。Further: the bile acid is added to a diet containing rice bran oil and / or full fat soybean meal.
进一步的：所述胆汁酸的添加量为日粮总质量的0.03％～0.1％。Further: the added amount of the bile acid is 0.03% to 0.1% of the total mass of the diet.
进一步的：所述米糠油用量占日粮总量的质量比为0.7％，断奶仔猪日增重最高且料肉比最低。Further: the mass ratio of the amount of rice bran oil to the total diet is 0.7%, the daily weight gain of weaned piglets is the highest, and the feed-to-meat ratio is the lowest.
本发明还提供了胆汁酸在制备用于牛蛙的饲料添加剂中的应用。The invention also provides the application of bile acid in the preparation of feed additive for bullfrog.
所述饲料中添加100mg/kg-200mg/kg胆汁酸。100 mg / kg-200 mg / kg bile acid is added to the feed.
本发明还提供了胆汁酸在制备用于大口黑鲈的饲料添加剂中的应用。The invention also provides the application of bile acid in the preparation of feed additives for largemouth bass.
所述大口黑鲈饲料中胆汁酸的添加量为200mg/kg-450mg/kg。The added amount of bile acid in the largemouth bass feed is 200mg / kg-450mg / kg.
与现有技术相比，本发明的优点和技术效果是：本发明通过实验证明将胆汁酸添加到断奶仔猪日粮中，可提高其生长性能，提高营养物质特别是脂肪的消化率，降低植物蛋白的抗营养因子，从而全面改善仔猪生长性能，提高饲料利用率和末重均匀度，降低造肉成本，提高经济效益回报。Compared with the prior art, the advantages and technical effects of the present invention are: The present invention proves through experiments that adding bile acid to the diet of weaned piglets can improve its growth performance, improve the digestibility of nutrients, especially fat, and reduce the plant Anti-nutritional factors of protein, thus comprehensively improve the growth performance of piglets, improve feed utilization and uniformity of final weight, reduce meat production costs, and improve economic returns.
本发明通过实验证明，饲料中添加胆汁酸可提高牛蛙肠道蛋白酶和脂肪酶活力，促进饲料营养的消化吸收，提高牛蛙对饲料干物质、蛋白质和脂肪的表观消化率，从而提高了牛蛙的增重率和饲料效率。饲料中添加胆汁酸可降低牛蛙机体蛋白质的分解代谢，促进蛋白质的沉积和脂肪分解，从而降低脏体比，提高牛蛙可食比例，增加机体蛋白质含量。饲料中添加胆汁酸可降低牛蛙肝脏脂肪水平和丙二醛含量，使肝脏处于低氧化应激水平，从而维护健康的肝脏组织结构，节约了抗氧化的能量消耗。The invention proves through experiments that the addition of bile acid to the feed can increase the intestinal protease and lipase activities of the bullfrog, promote the digestion and absorption of feed nutrients, and increase the apparent digestibility of the bullfrog to dry matter, protein and fat in the feed, thereby improving Weight gain and feed efficiency. Adding bile acid to the feed can reduce the catabolism of bullfrog body protein, promote protein deposition and lipolysis, thereby reducing the ratio of visceral body, increasing the edible proportion of bullfrog, and increasing the body protein content. Adding bile acid to the feed can reduce the fat level and malondialdehyde content of the bullfrog liver, keep the liver at a low oxidative stress level, thereby maintaining healthy liver tissue structure and saving antioxidant energy expenditure.
本发明通过试验结果证明饲料中添加适量胆汁酸可有效促进摄食和生长性能，方差分析结果显示饲料中添加300mg/kg胆汁酸，大口黑鲈获得最高生长性能，且肝脏胆汁酸含量与D240组没有差异。在以上添加范围内，胆汁酸有效降低大口黑鲈肝脏中MDA含量，提高抗氧化能力；通过提高血浆中HDL-C含量显著促进脂肪代谢，同时有效提高了肠道淀粉酶和脂 肪酶活性。本发明通过实验证明了将胆汁酸添加在大口黑鲈饲料中的切实可行性，具有良好的市场应用前景。The experiment proves that adding appropriate amount of bile acid to the feed can effectively promote feeding and growth performance. The analysis of variance shows that adding 300 mg / kg bile acid to the feed, large mouth bass has the highest growth performance, and the liver bile acid content is not the same as that of the D240 group. difference. Within the above range of addition, bile acid effectively reduces the MDA content in the liver of largemouth bass and improves the antioxidant capacity; by increasing the HDL-C content in the plasma, it significantly promotes fat metabolism and effectively improves the intestinal amylase and lipase activities. The invention proves the feasibility of adding bile acid to the largemouth bass feed through experiments, and has a good market application prospect.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是饲料中添加100mg/kg胆汁酸处理后的牛蛙肝脏组织切片。Figure 1 is a section of bullfrog liver tissue treated with 100 mg / kg bile acid added to the feed.
图2是饲料中添加200mg/kg胆汁酸处理后的牛蛙肝脏组织切片。Figure 2 is a section of bullfrog liver tissue treated with 200 mg / kg bile acid added to the feed.
图3是饲料中添加300mg/kg胆汁酸处理后的牛蛙肝脏组织切片。Figure 3 is a section of bullfrog liver tissue treated with 300 mg / kg bile acid added to the feed.
图4是饲料中添加400mg/kg胆汁酸处理后的牛蛙肝脏组织切片。Figure 4 is a section of bullfrog liver tissue treated with 400 mg / kg bile acid added to the feed.
图5表示胆汁酸含量对大口黑鲈特定生长率的影响关系；Figure 5 shows the effect of bile acid content on the specific growth rate of largemouth bass;
图6表示饲料中胆汁酸含量对大口黑鲈摄食率的影响关系；Figure 6 shows the effect of bile acid content on the feeding rate of largemouth bass;
图7表示饲料中胆汁酸含量对大口黑鲈肝脏中总胆汁酸含量的影响关系。Figure 7 shows the effect of bile acid content in feed on total bile acid content in the liver of largemouth bass.
具体实施方式detailed description
下面结合具体实施例对本发明的技术方案做进一步详细的描述。The technical solution of the present invention is described in further detail below with reference to specific embodiments.
实施例1Example 1
与成年畜禽相比，幼龄畜禽对脂肪的消化率较低。本发明为了提高断奶仔猪的生长性能，进行了以下试验。本发明试验的宗旨在于评估在含有米糠油、全脂豆粕和二者混合物的日粮中添加胆汁酸对仔猪生长性能的影响。Compared with adult animals, young animals and poultry have lower fat digestibility. In order to improve the growth performance of weaned piglets, the present invention performs the following tests. The purpose of the test of the present invention is to evaluate the effect of adding bile acids to diets containing rice bran oil, whole fat soybean meal, and a mixture of the two on the growth performance of piglets.
本发明所述的胆汁酸经过皂化、脱色、酸化、纯化和重结晶五个步骤制得，其详细步骤如下：The bile acid according to the present invention is prepared through five steps of saponification, decolorization, acidification, purification and recrystallization. The detailed steps are as follows:
1、皂化1.Saponification
称取粉碎的胆膏200kg，加入R201反应釜中，按照固液比1∶10加入10％的氢氧化钠溶液2000L。待R201反应釜中的溶液升温至100℃时开始计时，保持沸腾状态12h。将皂化液冷却至固液分层，用管道离心泵将上清液打至储槽中，反应釜中的剩余固体即为胆膏皂化品。200 kg of crushed bile was weighed, added to an R201 reaction kettle, and 2000 L of a 10% sodium hydroxide solution was added at a solid-liquid ratio of 1:10. When the temperature of the solution in the R201 reaction kettle was raised to 100 ° C, the timer was started, and the boiling state was maintained for 12 hours. The saponified liquid is cooled to a solid-liquid layer, and the supernatant liquid is blown into a storage tank with a pipeline centrifugal pump. The remaining solid in the reaction kettle is a bile saponified product.
2、脱色2.Dyeing
向反应釜中加1200kg自来水加热至80℃，使胆膏皂化品完全溶解(加水体积：胆膏质量＝6∶1)。然后加入60L双氧水，搅拌均匀后，在80℃下反应2h。反应完毕后，将溶液经管道中的过滤网装置过滤至酸化罐中。管道中的过滤网可将皂化后产生的絮状杂质过滤掉。Add 1200 kg of tap water to the reaction kettle and heat to 80 ° C to completely dissolve the bile saponified product (water volume: bile mass = 6: 1). Then, 60 L of hydrogen peroxide was added, and after stirring, the reaction was performed at 80 ° C. for 2 h. After the reaction is completed, the solution is filtered into the acidification tank through a filter device in the pipeline. The filter in the pipe can filter out the flocculent impurities generated after saponification.
3、酸化3.Acidification
通过管道离心泵打至酸化罐中之后，在罐体夹层中通冷凝水将溶液冷却至室温。通过酸计量罐往酸化槽中缓慢加入5％的盐酸溶液，不断搅拌。待溶液pH值在3左右时，停止加酸，此时得到白色固形物。After being driven into the acidification tank through a pipeline centrifugal pump, the solution was cooled to room temperature by passing condensed water through the interlayer of the tank body. Slowly add a 5% hydrochloric acid solution to the acidification tank through the acid metering tank and keep stirring. When the pH of the solution is around 3, acid addition is stopped, and a white solid is obtained at this time.
4、纯化4.Purification
将板框过滤机连接至酸化罐上。将白色固形物用板框过滤机过滤。过滤后，加水冲洗，待过滤出的水检测呈中性为水洗结束。水洗的目的是将附着在胆汁酸固形物表面的水溶性杂质去除。将过滤后的白色固形物放入烘箱中，在100℃下干燥至水分不高于10％，得胆汁酸粗品。Connect the plate and frame filter to the acidification tank. The white solid was filtered with a plate and frame filter. After filtering, rinse with water. The water to be filtered out is neutral and the washing is finished. The purpose of water washing is to remove water-soluble impurities attached to the surface of bile acid solids. The filtered white solid was placed in an oven and dried at 100 ° C to a moisture content of no more than 10% to obtain a crude bile acid.
5、重结晶5.Recrystallization
将得到的胆汁酸粗品投入萃取罐中，加入乙酸乙酯(加入乙酸乙酯的体积与胆汁酸粗品质量之比为10∶1)，搅拌溶解，待胆汁酸粗品基本溶解，加入无水硫酸钠脱水，收集溶液。将溶液打入浓缩罐中进行减压浓缩，回收乙酸乙酯。将浓缩液用压滤机压滤得到胆汁酸。Put the obtained crude bile acid into an extraction tank, add ethyl acetate (the ratio of the volume of ethyl acetate to the crude quality of bile acid is 10: 1), and stir to dissolve. After the crude bile acid is basically dissolved, add anhydrous sodium sulfate. Dehydrate and collect the solution. The solution was poured into a concentration tank and concentrated under reduced pressure, and ethyl acetate was recovered. The concentrated solution was filtered with a filter press to obtain bile acid.
6、干燥6.Drying
将得到的胆汁酸平铺在不锈钢烘干盘中，放入烘箱，通蒸汽进行真空干燥，干燥温度为100℃。待胆汁酸变成白色粉末状时，取样检测，如水分不超过1％，则视为干燥完毕。The obtained bile acid was tiled in a stainless steel drying dish, put into an oven, and vacuum-dried with steam, and the drying temperature was 100 ° C. When the bile acid turns into a white powder, sampling and testing, if the moisture does not exceed 1%, it is considered to be dry.
实施例2Example 2
1.材料与方法1. Materials and methods
试验在泰国孔敬省的一所管理规范的商业猪场(Reang Sifi Farm)进行。480头28日龄断奶三元杂交(杜洛克×大约克×长白)仔猪(均重8.62-8.65kg)分成4个处理，每个处理4个重复，每个重复30头，公母各半。仔猪饲养在可喷雾调节温度的保育舍中，试验期为28-62日龄，自由采食和自由饮水。每舍有3个10孔的喂料器和4个***饮水器。称取试验仔猪始重和末重，每天记录采食量和死亡率至试验结束。仔猪均匀度为基于平均末重标准差2倍的百分数。试验组1(T1)含有米糠油和全脂大豆，不添加胆汁酸。另外三个试验组添加0.04％胆汁酸(胆汁酸占饲料总量的质量比为0.04％)，米糠油用量占饲料总量的质量比分别为0.7％，1.0％和1.3％，并以碎米补充(详见表1)。日粮以糊状饲喂。The trial was conducted at a well-regulated commercial pig farm (Reang Sifi Farm) in Khon Kaen Province, Thailand. 480 28-day-old weaned three-way crossbred (Dulock × about g × Changbai) piglets (average weight 8.62–8.65 kg) were divided into 4 treatments, each treatment was repeated 4 times, each 30 replicates, male and female half. The piglets were housed in a nursery house with spray-adjustable temperature, and the test period was 28-62 days old, with free feeding and drinking water. Each house has 3 10-hole feeders and 4 nipple drinkers. Weigh the start and end weight of the test piglets, and record the feed intake and mortality daily until the end of the test. Piglet uniformity is a percentage based on 2 times the standard deviation of the mean final weight. Test group 1 (T1) contained rice bran oil and full-fat soybeans, and no bile acid was added. The other three test groups were supplemented with 0.04% bile acid (the mass ratio of bile acid to the total feed was 0.04%), and the mass ratio of rice bran oil to the total feed was 0.7%, 1.0% and 1.3%, respectively, and crushed rice Supplement (see Table 1 for details). The diet was fed in a paste form.
2.统计分析2. Statistical analysis
所有反应变量数据采用SAS软件中通用线性模型的单因素方差分析，组间差异采用邓肯多重比较进行处理。显著性分析均基于0.05的概率。All response variables were analyzed by one-way analysis of variance using the universal linear model in SAS software. Differences between groups were processed using Duncan multiple comparisons. The significance analysis was based on a probability of 0.05.
3.实验结果3. Experimental results
胆汁酸对仔猪生长性能的影响试验结果见表2，在整个试验期间，试验组日粮添加0.04％胆汁酸，米糠油用量为0.70％，1.00％和1.3％，试验期34天，添加胆汁酸组的仔猪体重(BW)、体增重(BWG，末重-初重)、采食量(FI)、料肉比(FCR采食量/末重-初重)、成活率(SR，活仔数/实验组头数)、末重均匀度(UBWT，平均体重±10％的个体数/样本总数)和造肉成本(FCG，饲养成本/(末重-初重))均优于或接近对照组。与对照组相比，仔猪日粮中添加0.04％胆汁酸可明显改善生长性能，显著降低FCR(P＜0.05)；提高FI(P＞0.05)；改善UBWT(P＞0.05)；显著降低FCG(P＜0.05)。仔猪日粮中添加胆汁酸可提高净利润，其中添加0.7％米糠油的投资回报率(ROI)最高。The test results of the effects of bile acids on the growth performance of piglets are shown in Table 2. During the whole test period, the diet of the test group was supplemented with 0.04% bile acid, the amount of rice bran oil was 0.70%, 1.00% and 1.3%, and the test period was 34 days. Group piglets' body weight (BW), body weight gain (BWG, last weight-initial weight), feed intake (FI), feed-to-meat ratio (FCR feed intake / last weight-initial weight), survival rate (SR, number of live piglets) / Experimental group head number), uniformity of final weight (UBWT, number of individuals with average weight ± 10% / total number of samples) and meat production cost (FCG, rearing cost / (last weight-initial weight)) are better than or close to the control group . Compared with the control group, the addition of 0.04% bile acid to the piglet diet can significantly improve growth performance, significantly reduce FCR (P <0.05); increase FI (P> 0.05); improve UBWT (P> 0.05); significantly reduce FCG ( P <0.05). Addition of bile acids to the piglet diet can increase net profit, of which the addition of 0.7% rice bran oil has the highest return on investment (ROI).
据此结论：仔猪日粮中添加0.04％胆汁酸并添加0.7％米糠油可改善仔猪生长性能，提高BWG、SR和UBWT，降低FCR和FCG，提高ROI。According to this conclusion: adding 0.04% bile acid and 0.7% rice bran oil to the piglet diet can improve piglet growth performance, increase BWG, SR and UBWT, reduce FCR and FCG, and increase ROI.
与对照组相比，米糠油用量(0.70％，1.00％，1.30％)的3个试验组，添加0.04％的胆汁酸均可提高体重和体增重，但不同米糠油用量组之间差异不显著(P＞0.05)。显著降低料肉比，提高成活率，改善末重均匀度，但不同米糠油用量组之间差异不显著，显著降低造肉成本。Compared with the control group, the three test groups of rice bran oil dosage (0.70%, 1.00%, 1.30%), adding 0.04% bile acid can increase body weight and weight gain, but the difference between different rice bran oil dosage groups is not the same Significant (P> 0.05). Remarkably reduce the ratio of feed to meat, increase the survival rate, and improve the uniformity of the final weight, but the difference between the different rice bran oil dosage groups is not significant, which significantly reduces the cost of meat production.
本发明实验结果表明：添加胆汁酸可使仔猪对饲料的消化吸收达到最佳，其中降低0.7％米糠油的试验组日增重最高，料肉比最低，经济效益回报最高。结果亦表明：添加胆汁酸的3个试验组经济效益回报均高于对照组，并且降低0.7％米糠油的试验组的投资回报最高。The experimental results of the present invention show that adding bile acid can optimize the digestion and absorption of feed for piglets, in which the experimental group that reduced 0.7% rice bran oil has the highest daily weight gain, the lowest feed-to-meat ratio, and the highest economic benefit return. The results also showed that the return of economic benefits of the three experimental groups added with bile acid was higher than that of the control group, and the experimental group that reduced 0.7% rice bran oil had the highest return on investment.
表1供试断奶仔猪日粮组成Table 1 Diet composition for test weaned piglets
Figure PCTCN2019095091-appb-000001
Figure PCTCN2019095091-appb-000001
本发明中断奶仔猪所使用的每公斤日粮中含有：维生素A：2500IU.；维生素D 3：250IU；维生素E：20IU；维生素B 12：0.2mg；泛酸：12mg；烟酸：20mg；核黄素：4mg；硫胺素：2mg；氯化胆碱1g；维生素B 6：2mg；生物素：0.3mg；维生素K 3：0.5mg；叶酸：0.3mg；乙氧基喹啉：12.5mg；铜：250mg；碘：0.4mg；铁：100mg；锰：40mg；硒：0.3mg；锌：100mg；钴：0.14mg；trimulin(一种防腹泻抗生素)：100mg；阿莫西林：300mg；抗敌素：200mg；金霉素：400mg。 Weaned present invention used per kg diet contains: vitamin A: 2500IU .; Vitamin D 3: 250IU; Vitamin E: 20IU; Vitamin B 12: 0.2mg; pantothenic acid: 12 mg of; Niacin: 20mg; riboflavin Su: 4mg; thiamine: 2mg; choline chloride 1g; vitamin B 6: 2mg; biotin: 0.3mg; vitamin K 3: 0.5mg; folic acid: 0.3mg; ethoxyquin: 12.5mg; copper : 250mg; Iodine: 0.4mg; Iron: 100mg; Manganese: 40mg; Selenium: 0.3mg; Zinc: 100mg; Cobalt: 0.14mg; : 200 mg; chlortetracycline: 400 mg.
表2日粮中添加胆汁酸对断奶仔猪生长性能的影响(28-62日龄)Table 2 Effect of adding bile acid to the diet on growth performance of weaned piglets (28-62 days of age)
Figure PCTCN2019095091-appb-000002
Figure PCTCN2019095091-appb-000002
Figure PCTCN2019095091-appb-000003
Figure PCTCN2019095091-appb-000003
注：同行平均值上标字母不同者表示差异显著(p＜0.05)Note: The difference between the superscript letters of the peer average means that the difference is significant (p <0.05)
平均每头净利润＝体增重×单价(82泰铢/kg)-饲料成本(泰铢/头)Average net profit per head = weight gain × unit price (82 baht / kg)-feed cost (baht / head)
本发明通过试验结果表明：与不添加胆汁酸的对照组相比，试验期(34d)内，断奶仔猪日粮全程添加胆汁酸可提高其生长性能，其中添加0.7％的米糠油的试验组日增重最高，料肉比最低，经济效益回报最高。本发明在断奶仔猪日粮全程添加胆汁酸，能够提高营养物质特别是脂肪的消化率，降低植物蛋白的抗营养因子，从而全面改善仔猪生长性能，提高饲料利用率和末重均匀度，降低造肉成本，提高经济效益回报。The test results of the present invention show that compared with the control group without added bile acid, the growth performance of the weaned piglets during the whole period of the experimental period (34d) can be improved by adding bile acid, and the test group added 0.7% rice bran oil daily. The highest weight gain, the lowest meat-to-meat ratio, and the highest economic returns. The present invention adds bile acid to the diet of weaned piglets, which can improve the digestibility of nutrients, especially fat, and reduce the anti-nutritional factors of plant proteins, thereby comprehensively improving the growth performance of piglets, increasing the utilization rate of feed and the uniformity of final weight, reducing the production Meat costs increase economic returns.
实施例3Example 3
1.材料与方法1. Materials and methods
1.1实验基础饲料1.1 Experimental basic feed
以鱼粉和豆粕为主要蛋白源，棕榈油主要脂肪源配制出基础饲料(粗蛋白40.8％，粗脂肪7％)。分别在每千克基础饲料中添加0、100、200和300mg胆汁酸(含有猪胆酸10％、猪去氧胆酸67％、鹅去氧胆酸17％)配制出4种实验饲料，以面粉调平，在每种实验饲料中添加0.5％的Cr2O3作标记物。饲料原料经粉碎过60目筛，各原料按配比定量后混合均匀，加棕榈油，然后加入适量的水揉匀，经水产饲料膨化机制成直径为4.0mm的饲料颗粒，晾干储存在-20℃的冰箱中备用。Fish meal and soybean meal were used as the main protein sources, and palm oil was used as the main fat source to prepare a basic feed (crude protein 40.8%, crude fat 7%). Add 0, 100, 200, and 300 mg of bile acid (contains 10% of cholic acid, 67% of hyodeoxycholic acid, and 17% of chenodeoxycholic acid) to each kilogram of basic feed to prepare 4 experimental feeds. In addition, 0.5% Cr2O3 was added as a marker in each experimental feed. The feed ingredients are pulverized through a 60-mesh sieve. After the raw materials are quantified according to the ratio, they are mixed uniformly, and palm oil is added, and then an appropriate amount of water is added to knead them. Store in refrigerator at ℃.
1.2动物饲养与管理1.2 Animal Breeding and Management
实验用蛙购于厦门市同安区一养殖场的同一批培育的牛蛙。正式实验前，牛蛙放于室内暂养缸中暂养，以实验对照饲料饱食投喂，使之逐渐适应实验饲料及养殖环境。暂养15d后，将牛蛙饥饿24h，挑选个体大小均匀的健康牛蛙随机分配到室内养殖***中的12只水族缸(150L)中进行养殖实验。The experimental frogs were purchased from the same batch of bullfrogs cultivated in a farm in Tongan District, Xiamen. Before the formal experiment, the bullfrog was kept in an indoor holding tank and fed with experimental control feed to make it gradually adapt to the experimental feed and breeding environment. After 15 days of temporary breeding, the bullfrogs were starved for 24 hours, and healthy bullfrogs of uniform size were randomly allocated to 12 aquariums (150L) in the indoor breeding system for breeding experiments.
实验牛蛙初始体重为(75±4.23)g，随机分为4组，每组3个重复，每个重复放养12只牛蛙。养殖实验持续8周。根据每天各缸牛蛙的摄食情况进行适当调整达到饱食投喂，每天投喂2次(8：00，18：00)，投喂30分钟前对各缸进行换水和清洗。养殖期间记录牛蛙的摄食和死亡情况。The initial weight of the experimental bullfrog was (75 ± 4.23) g, which was randomly divided into 4 groups, each group had 3 repetitions, and each restocked 12 bullfrogs. Farming experiments lasted 8 weeks. According to the feeding situation of bullfrogs in each tank, adjust to achieve full feeding. Feed 2 times a day (8:00, 18:00), and change the water and clean the tanks 30 minutes before feeding. Feeding and death of bullfrogs were recorded during breeding.
1.3牛蛙消化率实验1.3 Bullfrog digestibility experiment
养殖实验结束后，对各缸剩余牛蛙进行消化率实验。采用网箱(为底面积略小于缸底的木质网箱，顶面覆盖密网，高约6cm)收集粪便。下午投喂2小时后，清洗缸内残料和*** 物，铺设网箱，于次日早上投喂前取出网箱，收集粪便，置-20℃冰箱以备分析。为减少处理应激，维持牛蛙正常生长，粪便每隔一天收集一次，直至收集够量为止。After the breeding experiment was over, digestion experiments were performed on the remaining bullfrogs in each tank. Net cages (wooden cages with a bottom area slightly smaller than the bottom of the tank, top surface covered with dense nets, about 6 cm high) were used to collect feces. After 2 hours of feeding in the afternoon, clean the remnants and excreta in the tank, lay the cage, remove the cage before feeding the next morning, collect the feces, and place it in a -20 ° C refrigerator for analysis. In order to reduce the treatment stress and maintain the normal growth of bullfrogs, the feces were collected every other day until a sufficient amount was collected.
1.5牛蛙肝脏病理切片1.5 Bullfrog liver pathological section
Bouin氏固定液固定牛蛙肝脏，用常规石蜡切片法切片，切片厚度7～9μm，HE染色。在Nikon eslipse 50i显微镜下观察，并用Nikon coolpix 5400数码相机照相。Bouin's fixative was used to fix the bullfrog liver, and the sections were sliced by conventional paraffin sectioning with a thickness of 7-9 μm, and stained with HE. Observe under a Nikon eslipse 50i microscope and take pictures with a Nikon Coolpix 5400 digital camera.
2.结果2. Results
2.1饲料中添加胆汁酸对牛蛙生长性能的影响2.1 Effect of adding bile acid to feed on growth performance of bullfrog
经8周养殖实验，饲料中添加胆汁酸对牛蛙生长性能的影响见表3。胆汁酸为200mg/kg添加组牛蛙增重率、特定生长率、蛋白质效率和饲料效率最高，分别为220.95％，1.91，1.09，3.26，较对照组分别提高了10.35％，15.96％，13.76％，13.59％，各组摄食率无显著性差异；添加100和300mg/kg胆汁酸对牛蛙增重率无显著影响(P＞0.05)；各处理组成活率无显著性差异(P＞0.05)。After 8 weeks of breeding experiments, the effect of adding bile acid to the feed on the growth performance of bullfrogs is shown in Table 3. The weight increase rate, specific growth rate, protein efficiency, and feed efficiency of bullfrogs in the bile acid addition group of 200mg / kg were the highest, being 220.95%, 1.91, 1.09, and 3.26, which were 10.35%, 15.96%, and 13.76% higher than the control group, respectively. 13.59%, no significant difference in feeding rate between groups; the addition of bile acid at 100 and 300 mg / kg had no significant effect on the rate of weight gain of bullfrogs (P> 0.05); there was no significant difference in the survival rate of each treatment composition (P> 0.05).
表3饲料中添加胆汁酸对牛蛙生长性能的影响Table 3 Effect of adding bile acid to feed on growth performance of bullfrog
Figure PCTCN2019095091-appb-000004
Figure PCTCN2019095091-appb-000004
注：同行上标小写字母不同代表差异显著(P＜0.05)，以下各表相同。Note: Different uppercase and lowercase letters of the peers represent significant differences (P <0.05). The following tables are the same.
2.2饲料中添加胆汁酸对牛蛙内脏指数的影响2.2 Effect of adding bile acid to feed on bullfrog visceral index
饲料中添加200mg/kg胆汁酸，显著降低了牛蛙的脏体指数(P＜0.05)，添加量为100mg/kg和300mg/kg的各组均较对照组有所下降(P＞0.05)。各添加组的肝体指数无显著性差异(P＞0.05)(表4)。Adding 200mg / kg bile acid to the feed significantly reduced the bullfrog's visceral index (P <0.05), and the groups at 100mg / kg and 300mg / kg decreased compared with the control group (P> 0.05). There was no significant difference in liver body index among the added groups (P> 0.05) (Table 4).
表4饲料中添加胆汁酸对牛蛙内脏指数的影响％Table 4 Effect of adding bile acid to feed on bullfrog visceral index%
Figure PCTCN2019095091-appb-000005
Figure PCTCN2019095091-appb-000005
2.3饲料中添加胆汁酸对牛蛙全体和肌肉营养成分的影响2.3 Effect of adding bile acid in feed on nutritional composition of whole bullfrog and muscle
牛蛙全体、肌肉和肝脏成分见表5。各组牛蛙全体的水分和粗灰分含量无显著差异(P＞0.05)，但胆汁酸添加组牛蛙全体粗脂肪含量显著低于对照组，当胆汁酸添加量为100，200 和300mg/kg时，全体粗脂肪含量较对照组分别降低12.69％，13.46％，15.38％(P＜0.05)；随着胆汁酸的添加量增加，粗蛋白含量较对照组均有提高，其中当胆汁酸添加量为200mg/kg时，全体粗蛋白含量显著高于对照组(P＜0.05)。The bullfrog overall, muscle and liver components are shown in Table 5. There was no significant difference in the water and crude ash content of the bullfrogs in each group (P> 0.05), but the crude fat content of the bullfrogs in the bile acid addition group was significantly lower than that in the control group. When the bile acid addition amount was 100, 200, and 300 mg / kg, Compared with the control group, the total crude fat content was reduced by 12.69%, 13.46%, and 15.38% (P <0.05). With the increase of the amount of bile acid, the content of crude protein increased compared with the control group. When the amount of bile acid was 200 mg At kg / kg, the total crude protein content was significantly higher than the control group (P <0.05).
肌肉中水分、粗脂肪、粗灰分含量无显著性差异(P＞0.05)。饲料中添加胆汁酸的各处理组粗蛋白含量显著高于对照组(P＜0.05)，其中胆汁酸添加量为200mg/kg时最高。There were no significant differences in the contents of water, crude fat and crude ash (P> 0.05). The content of crude protein in the treatment groups supplemented with bile acid was significantly higher than that in the control group (P <0.05), and the highest was when the amount of bile acid was 200 mg / kg.
肝脏中水分和粗灰分含量均无显著性差异(P＞0.05)。当添加100和200mg/kg胆汁酸后，肝脏中粗蛋白含量显著高于对照组(P＜0.05)；与对照组比较，在胆汁酸添加量为100mg/kg时显著降低了肝脏粗脂肪含量(P＜0.05)。There were no significant differences in the contents of water and crude ash in the liver (P> 0.05). When 100 and 200 mg / kg bile acid were added, the crude protein content in the liver was significantly higher than that in the control group (P <0.05); compared with the control group, the crude fat content in the liver was significantly reduced when the bile acid was added at 100 mg / kg ( P <0.05).
表5饲料中添加胆汁酸对牛蛙全体和肌肉组成成分的影响％Table 5 Effect of adding bile acid to feed on overall and muscle composition of bullfrog
Figure PCTCN2019095091-appb-000006
Figure PCTCN2019095091-appb-000006
2.4饲料中添加胆汁酸对血清生化指标的影响2.4 Effect of adding bile acid to feed on serum biochemical indicators
饲料中添加不同水平胆汁酸后，牛蛙血清胆固醇和血脂浓度较对照组均有上升的趋势，但差异不显著(P＞0.05)。饲料中添加胆汁酸对高密度脂蛋白胆固醇和低密度脂蛋白胆固醇含量无显著影响(P＞0.05)。当添加300mg/kg胆汁酸，尿素氮含量较对照组显著降低(P＜0.05)，添加100mg/kg胆汁酸时，血糖浓度较对照组显著降低(P＜0.05)。After adding different levels of bile acids to the feed, the serum cholesterol and blood lipid concentrations of bullfrogs increased compared with the control group, but the differences were not significant (P> 0.05). The addition of bile acids to feeds had no significant effect on the contents of HDL and LDL cholesterol (P> 0.05). When 300 mg / kg bile acid was added, the urea nitrogen content was significantly lower than that in the control group (P <0.05), and when 100 mg / kg bile acid was added, the blood glucose concentration was significantly lower than the control group (P <0.05).
表6饲料中添加胆汁酸对血清生化指标的影响(mmol/L)Table 6 Effect of adding bile acid to feed on serum biochemical indicators (mmol / L)
Figure PCTCN2019095091-appb-000007
Figure PCTCN2019095091-appb-000007
2.5饲料中添加胆汁酸对肠道蛋白酶、脂肪酶和淀粉酶的影响2.5 Effect of adding bile acid in feed on intestinal protease, lipase and amylase
与对照组相比，添加100mg/kg和200mg/kg胆汁酸均显著提高了肠道蛋白酶和脂肪酶活力(P＜0.05)，而添加量为300mg/kg时无显著性影响(P＞0.05)。各添加组的淀粉酶活力较对照组均有提高，但各组之间无显著性差异(P＞0.05)。Compared with the control group, the addition of bile acids at 100 mg / kg and 200 mg / kg significantly increased the intestinal protease and lipase activity (P <0.05), but there was no significant effect when added at 300 mg / kg (P> 0.05) . Compared with the control group, the amylase activity of each added group was increased, but there was no significant difference between the groups (P> 0.05).
表7饲料中添加胆汁酸对肠道蛋白酶，脂肪酶和淀粉酶活力的影响Table 7 Effect of adding bile acid to feed on intestinal protease, lipase and amylase activity
Figure PCTCN2019095091-appb-000008
Figure PCTCN2019095091-appb-000008
2.6牛蛙对添加胆汁酸饲料中营养物质表观消化率的影响2.6 Effect of Bullfrog on Apparent Digestibility of Nutrients in Bile Acid Feed
饲料中添加200mg/kg胆汁酸可显著提高牛蛙对饲料干物质、蛋白质和脂肪的表观消化率(P＜0.05)，而其他各胆汁酸添加组的消化率均高于对照组，但其差异未达显著水平(P＞0.05)。Adding 200mg / kg bile acid to the feed can significantly increase the apparent digestibility of dry matter, protein and fat of the bullfrog (P <0.05), while the digestibility of the other bile acid addition groups is higher than the control group, but the difference is Not significant level (P> 0.05).
表8牛蛙对添加胆汁酸饲料中粗蛋白和粗脂肪表观消化率的影响Table 8 Effect of Bullfrog on Apparent Digestibility of Crude Protein and Crude Fat in Bile Acid Supplement
Figure PCTCN2019095091-appb-000009
Figure PCTCN2019095091-appb-000009
2.7饲料中添加胆汁酸对牛蛙肝脏抗氧化酶的影响2.7 Effect of adding bile acid to feed on antioxidant activities of bullfrog liver
饲料中添加胆汁酸对肝脏SOD无显著性影响(P＞0.05)。添加100和300mg/kg胆汁酸显著降低了过氧化氢酶活力，添加量为200mg/kg时较对照组无显著变化，添加200和300mg/kg胆汁酸能显著降低MDA的活性(P＜0.05)。Addition of bile acid to the feed had no significant effect on SOD in the liver (P> 0.05). The addition of bile acid at 100 and 300 mg / kg significantly reduced the activity of catalase. When the amount of bile acid was 200 mg / kg, there was no significant change compared with the control group. The addition of bile acid at 200 and 300 mg / kg significantly reduced the activity of MDA (P <0.05). .
表9胆汁酸对肝脏抗氧化指标的影响Table 9 Effect of bile acids on liver antioxidant indexes
Figure PCTCN2019095091-appb-000010
Figure PCTCN2019095091-appb-000010
2.8饲料中添加胆汁酸对牛蛙肝脏代谢酶的影响2.8 Effect of Adding Bile Acid to Feed on Bullfrog Metabolic Enzymes
各组牛蛙肝脏谷草转氨酶和谷丙转氨酶活力无显著性差异(P＞0.05)。胆汁酸添加量为200mg/kg时，脂蛋白酯酶、肝酯酶和总酯酶活力均显著低于对照组(P＜0.05)，各添加组之间差异不显著(P＞0.05)。There was no significant difference in the activity of aspartate aminotransferase and alanine aminotransferase in the liver of bullfrog in each group (P> 0.05). When the amount of bile acid was 200 mg / kg, the activities of lipoprotein esterase, liver esterase and total esterase were significantly lower than those of the control group (P <0.05), and there was no significant difference between the added groups (P> 0.05).
表10胆汁酸对肝脏代谢指标的影响Table 10 Effect of bile acids on liver metabolic indexes
Figure PCTCN2019095091-appb-000011
Figure PCTCN2019095091-appb-000011
Figure PCTCN2019095091-appb-000012
Figure PCTCN2019095091-appb-000012
2.9饲料中添加胆汁酸对牛蛙肝脏组织结构的影响2.9 Effect of adding bile acid in feed on the liver tissue structure of bullfrog
实验结果如图1-4所示，通过实验结果可知，与对照组相比，饲料中分别添加胆汁酸100mg/kg、200mg/kg、300mg/kg、400mg/kg时，肝脏细胞排列较整齐，细胞间隙均一。The experimental results are shown in Figures 1-4. According to the experimental results, compared with the control group, when bile acids were added to the feed at 100 mg / kg, 200 mg / kg, 300 mg / kg, and 400 mg / kg, the liver cells were arranged neatly. The intercellular space is uniform.
综上，通过本发明的实验可以证明，饲料中添加胆汁酸可提高牛蛙肠道蛋白酶和脂肪酶活力，促进饲料营养的消化吸收，提高牛蛙对饲料干物质、蛋白质和脂肪的表观消化率，从而提高了牛蛙的增重率和饲料效率。In summary, through experiments of the present invention, it can be proved that the addition of bile acid to feed can increase the intestinal protease and lipase activity of bullfrogs, promote the digestion and absorption of feed nutrients, and increase the apparent digestibility of bullfrogs to feed dry matter, proteins and fats. This improves the weight gain rate and feed efficiency of bullfrogs.
饲料中添加胆汁酸可降低牛蛙机体蛋白质的分解代谢，促进蛋白质的沉积和脂肪分解，从而降低脏体比，提高牛蛙可食比例，增加机体蛋白质含量。Adding bile acid to the feed can reduce the catabolism of bullfrog body protein, promote protein deposition and lipolysis, thereby reducing the ratio of visceral body, increasing the edible proportion of bullfrog, and increasing the body protein content.
饲料中添加胆汁酸可降低牛蛙肝脏脂肪水平和丙二醛含量，使肝脏处于低氧化应激水平，从而维护健康的肝脏组织结构，节约了抗氧化的能量消耗。Adding bile acid to the feed can reduce the fat level and malondialdehyde content of the bullfrog liver, keep the liver at a low oxidative stress level, thereby maintaining healthy liver tissue structure and saving antioxidant energy expenditure.
根据本实验的结果，从牛蛙生长、饲料效率、体组成和肝脏健康角度考虑，建议牛蛙饲料中添加100-200mg/kg胆汁酸。According to the results of this experiment, from the perspective of bullfrog growth, feed efficiency, body composition and liver health, it is recommended to add 100-200mg / kg bile acid to the feed of bullfrog.
实施例4Example 4
1、材料与方法1. Materials and methods
1.1试验鱼1.1 Test fish
试验用水产靶动物为大口黑鲈(Micropterussalmoides)，购自佛山市三水白金水产种苗有限公司，试验正式开始前，试验鱼在养殖***中暂养2周，暂养期间投喂不含外源胆汁酸的基础饲料。The target animal used in the test was the largemouth bass (Micropterussalmoides), which was purchased from Foshan Sanshui Platinum Aquatic Seeds Co., Ltd. Before the test officially started, the test fish were temporarily kept in the breeding system for 2 weeks. Source of source bile acids.
治疗和预防措施(原因、时间、种类、持续时间等)：实验前用2％食盐水消毒实验鱼及养殖***。试验期间无疾病发生。Treatment and preventive measures (cause, time, type, duration, etc.): 2% saline solution was used to disinfect the experimental fish and the breeding system before the experiment. No disease occurred during the trial.
1.2受试物1.2 Test substance
试验所用胆汁酸包括猪胆酸、猪去氧胆酸和鹅去氧胆酸，本发明所述的胆汁酸经过皂化、脱色、酸化、纯化和重结晶五个步骤制得，其详细步骤如实施例1。The bile acids used in the test include pig bile acid, pig deoxycholic acid, and chenodeoxycholic acid. The bile acids described in the present invention are prepared through five steps: saponification, decolorization, acidification, purification, and recrystallization. The detailed steps are as in the examples. 1.
对胆汁酸的相关卫生指标进行了检测，受试物重金属含量均符合国家饲料卫生标准，结果见表11。The relevant sanitary indicators of bile acid were tested, and the heavy metal content of the tested substances all met the national feed hygiene standards. The results are shown in Table 11.
表11本发明所述受试物胆汁酸的卫生指标Table 11 Hygienic indicators of the bile acids of the test substance according to the present invention
Figure PCTCN2019095091-appb-000013
Figure PCTCN2019095091-appb-000013
1.3试验料1.3 Test material
在大口黑鲈的基础饲料中分别添加0、80、160、240、300、600mg/kg的胆汁酸，制成6种直径为2.0mm的膨化挤压浮性颗粒料，自然晾干后备用。6个处理组依次命名为D0、D80、 D160、D240、D300、D600。Bile acids of 0, 80, 160, 240, 300, and 600 mg / kg were added to the basic feed of largemouth bass, respectively, and 6 kinds of extruded and extruded floating pellets with a diameter of 2.0 mm were prepared, and then dried naturally for later use. The six processing groups are named D0, D80, D160, D240, D300, and D600 in this order.
饲料中的水分、粗蛋白质、粗脂肪、灰分和总能分别采用105℃常压干燥法、凯氏定氮法、酸水解全脂肪测定法、550℃灼烧法和氧弹仪燃烧法测定。饲料配方及营养成分分析(见表12)。Moisture, crude protein, crude fat, ash and total energy in feed were measured by 105 ℃ atmospheric pressure drying method, Kjeldahl method, acid hydrolysis total fat measurement method, 550 ℃ burning method and oxygen bomb combustion method. Feed formula and nutritional composition analysis (see Table 12).
表12.基础饲料配方及成分分析(风干基础，％)Table 12. Basic feed formula and analysis of ingredients (air-dried basis,%)
Figure PCTCN2019095091-appb-000014
Figure PCTCN2019095091-appb-000014
*胆汁酸在预混料中添加。* Bile acid is added to the premix.
1.4分组及试验管理1.4 Grouping and test management
试验在国家水产饲料安全评价基地(北京)室内循环流水养殖***中进行。随机挑选体质健康、个体均匀的大口黑鲈(起始平均体重6.17±0.02g)，分配到容积为0.26m的圆锥形养殖桶中。本试验设计5个处理组，每处理组6个重复，每桶30尾鱼。The trial was conducted in an indoor circulating flow aquaculture system of the National Aquatic Feed Safety Evaluation Base (Beijing). Largemouth bass with a healthy body and an even individual (starting average body weight 6.17 ± 0.02g) was randomly selected and allocated to a conical breeding barrel with a volume of 0.26m. This experiment was designed with 5 treatment groups, each with 6 repetitions and 30 fish per barrel.
每天表观饱食投喂2次，投喂时间分别为8：00，16：00。定期检测水质，水质条件保持在溶氧DO＞7.0mg/L，水温25±3℃；总氨氮NH 4 +-N＜0.3mg/L；pH＝7.5-8.5；水温25±3℃。 Apparently fed twice a day, feeding times were 8:00 and 16:00, respectively. Regularly check the water quality. The water quality conditions are maintained at DO DO 7.0mg / L, water temperature 25 ± 3 ℃; total ammonia nitrogen NH 4 + -N <0.3mg / L; pH = 7.5-8.5;
养殖试验从2014年7月14日到2014年9月21日，共70天。The breeding trials lasted for 70 days from July 14, 2014 to September 21, 2014.
2、评价指标2. Evaluation index
2.1生长指标2.1 Growth indicators
试验期间，记录每日摄食量，试验70天后，分别对各桶鱼称重并统计存活率饲料系数、增重率、特定生长率等。During the test, the daily food intake was recorded. After 70 days of the test, each barrel of fish was weighed and the survival rate feed coefficient, weight gain rate, specific growth rate, etc. were calculated.
各指标计算公式：Calculation formula of each indicator:
存活率(Survival rate，SR，％)＝100×终末鱼数量/初始鱼数量；Survival rate (Survival rate, SR,%) = 100 × number of terminal fish / number of initial fish;
相对增重率(Weight gain rate，WGR，％)＝100×(末鱼均重一初始鱼均重+死鱼重)/初始鱼均重；Relative weight gain rate (Weight gain rate, WGR,%) = 100 × (average weight of the last fish-average weight of the initial fish + weight of the dead fish) / average weight of the initial fish;
特定生长率率(Specific growth rate，SGR％/d)＝100×[n(FBW)-n(IBW)]/t；其中IBW为鱼体初始均重(g)；FBW为终末均重(g)；t为实验天数。Specific growth rate (Specific growth rate, SGR% / d) = 100 × [n (FBW) -n (IBW)] / t; where IBW is the initial average weight of the fish (g); FBW is the terminal average weight ( g); t is the number of experimental days.
饲料系数(Feed conversion ratio，FCR)＝摄食量/(末鱼重+死鱼重一初始鱼重)；Feed coefficient (FCR) = food intake / (last fish weight + dead fish weight-initial fish weight);
摄食率(Feeding rate，FR％body weight/d)＝100×C/((W0+Wt+Wd)/2)/t，其中W0为鱼体初始总重(g)，Wt为终末总重(g)，Wd为死亡鱼体总重(g)；C为摄食量(g)，t为试验天数。Feeding rate (FR% body weight / d) = 100 × C / ((W0 + Wt + Wd) / 2) / t, where W0 is the initial total weight of the fish (g) and Wt is the total final weight (g), Wd is the total weight of dead fish (g); C is the food intake (g), and t is the number of test days.
2.2脏器指数2.2 Organ index
每桶随机取3尾鱼测量体长、体重、内脏重、肝脏重、脾脏重。Three fish were randomly selected from each barrel to measure body length, body weight, internal organ weight, liver weight, and spleen weight.
各指标计算公式：Calculation formula of each indicator:
肥满度(Condition factor，CF，g/cm3)＝平均体重(g)/[平均体长(cm)] 3； Fullness (Condition factor, CF, g / cm3) = average weight (g) / [average body length (cm)] 3 ;
肝体比(Hepatosomatic index，HSI，％)＝100×肝脏重/全鱼重；Hepatosomal ratio (Hepatosomatic index, HSI,%) = 100 × liver weight / whole fish weight;
脏体比(Viscerasomatic index，VSI％)＝100×内脏重/体重；Viscerasomatic index (VSI%) = 100 × visceral weight / body weight;
脾脏指数(Spleen index，SSI，％)＝100×脾脏重/全鱼重；Spleen index (Spleen index, SSI,%) = 100 × spleen weight / whole fish weight;
2.3血浆脂肪代谢指标及抗氧化指标2.3 Plasma fat metabolism index and antioxidant index
每桶随机取3尾鱼，三氯叔丁醇麻醉后尾静脉取血，加入抗凝剂(2％NaF+4％草酸钾)，用量为30ul/ml血液。将抗凝血在4℃，400omp的条件下离心10min，取上清血浆保存于-80℃的冰箱中待测。检测指标有：血浆脂肪代谢指标：总胆固醇(TC)、甘油三酯(TG)、总胆红素(TBILI)、高密度脂蛋白(IHDL)、碱性磷酸酶(ALP)；血浆抗氧化指标：丙二醛(MDA)、总超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)、谷胱甘肽-S转移酶每(GST)、谷胱甘肽过氧化物酶(GSHI-PX)等。Three fish were randomly collected per barrel. Blood was collected from the tail vein after anesthesia with trichlorobutanol, and an anticoagulant (2% NaF + 4% potassium oxalate) was added in an amount of 30 ul / ml of blood. The anticoagulated blood was centrifuged at 400 ° C. for 10 min at 4 ° C., and the supernatant plasma was stored in a refrigerator at −80 ° C. to be tested. Detection indicators are: plasma lipid metabolism indicators: total cholesterol (TC), triglycerides (TG), total bilirubin (TBILI), high density lipoprotein (IHDL), alkaline phosphatase (ALP); plasma antioxidant indicators : Malondialdehyde (MDA), total superoxide dismutase (SOD), total antioxidant capacity (T-AOC), glutathione-S transferase per (GST), glutathione peroxidase ( GSHI-PX) and so on.
2.4肝脏抗氧化指标、淀粉酶、脂肪酶及胆汁酸含量2.4 Liver antioxidant index, amylase, lipase and bile acid content
每桶随机取2尾鱼的肝脏，保存于-80℃的冰箱中。The livers of 2 fishes were randomly collected from each bucket and stored in a refrigerator at -80 ° C.
肝脏组织样品制备：取组织03-0.5g，加入介质匀浆(匀浆介质根据检测指标确定)，组织充分粉碎混匀后，3000mp，离心10分钟，取上清液，进行检测。Preparation of liver tissue samples: Take 03-0.5g of tissue, add media to homogenize (the homogenized medium is determined according to the detection index), and after the tissue is fully crushed and mixed, centrifuge at 3000 mp for 10 minutes, and take the supernatant for testing.
2.5肠道消化酶活性2.5 Intestinal digestive enzyme activity
每桶随机取2尾鱼的中肠和后肠(取中部靠中间1cm长作为中肠，取后一部分靠近***处1cm作为后肠)，保存于-80℃的冰箱中肠道组织样品制备；取组织0.2-0.5g，加入介质匀浆(胆汁酸匀浆介质为无水乙醇，淀粉酶及脂肪酶匀浆介质为0.7％6的生理盐水)，组织充分粉碎混匀后3000转/分钟，离心10分钟，取上清液，用用于检测淀粉酶、脂肪酶活性。Take the fish's midgut and hindgut with 2 tails randomly (take the middle part 1cm long as the midgut, and take the latter part 1cm close to the anus as the hindgut), and store it in -80 ℃ refrigerator midgut tissue samples; Take 0.2-0.5g of tissue, add media homogenate (the bile acid homogenization medium is absolute ethanol, amylase and lipase homogenization medium is 0.7% 6 physiological saline), and the tissue is fully crushed and mixed at 3000 rpm, After centrifugation for 10 minutes, the supernatant was taken and used for detecting amylase and lipase activity.
2.6胆囊胆汁酸含量2.6 Gallbladder Bile Acid Content
每桶随机取2尾鱼的胆囊的胆汁，保存于-80℃的冰箱中胆汁样品制备：取取出胆汁，加入无水乙醇匀浆，充分混匀后，300omp，离心10分钟，取上清液，进行检测。本试验所用试剂盒均购自南京建成生物工程研究所。Collect bile from the gallbladder of 2 fish at random in each bucket, and store the bile sample in a refrigerator at -80 ° C. Take the bile out, add absolute ethanol, and homogenize it thoroughly. Centrifuge at 300omp for 10 minutes and take the supernatant. To test. The kits used in this test were purchased from Nanjing Jiancheng Biotechnology Research Institute.
3、数据统计3. Statistics
试验数据以平均数士标准误(Mean±S.E.)表示，所有数据用SPSS Statistics17.0软件进行单因子方差分析(ANOVA)，Duncan′s多重比较检验差异的显著性，显著性水平以P＜0.05计。The test data were expressed as mean ± standard error (Mean ± SE). All data were analyzed by one-way analysis of variance (ANOVA) using SPSS Statistics 17.0 software. Duncan's multiple comparison test was used to test the significance of the differences. The significance level was P <0.05. meter.
4、试验结果4. Test results
4.1饲料中添加不同剂量的胆汁酸对大口黑鲈生长性能的影响4.1 Effect of adding different doses of bile acids to the growth performance of largemouth bass
在饲料中添加胆汁酸对大口黑鲈生长性能的影响见表13。各组大口黑鲈存活率均高于96.67％，且各组没有显著差异。D300组末均重显著高于其他组，其WGR和SGR亦达到最高，并显著高于对照组和D80，D160组。该组摄食率也达到最高水平，并显著高于对照组(P＜0.05)。各组饲料系数没有显著差异(P＞-0.05)。依据特定生长率和摄食率结果进行回归模型分析，获得饲料中胆汁酸最适添加量为440.5mg/kg和283.3mg/kg(图5和图6)。The effects of adding bile acids to the feed on the growth performance of largemouth bass are shown in Table 13. The survival rate of largemouth bass was higher than 96.67% in each group, and there was no significant difference between the groups. The end weight of D300 group was significantly higher than that of other groups, and its WGR and SGR also reached the highest, and were significantly higher than those of control group and D80 and D160 groups. The feeding rate of this group also reached the highest level, and was significantly higher than that of the control group (P <0.05). There were no significant differences in feed coefficients among the groups (P> -0.05). Based on the results of specific growth rate and feeding rate, regression model analysis was performed, and the optimal amounts of bile acids in the feed were 440.5 mg / kg and 283.3 mg / kg (Figures 5 and 6).
表13不同剂量胆汁酸对大口黑鲈生长性能的影响(Mean±S.E.)Table 13 Effect of different doses of bile acids on growth performance of largemouth bass (Mean ± S.E.)
Figure PCTCN2019095091-appb-000015
Figure PCTCN2019095091-appb-000015
4.2饲料中添加不同剂量的胆汁酸对大口黑鲈形体指标的影响4.2 Effects of adding different doses of bile acids to the body index of largemouth bass
饲料中添加胆汁酸对大口黑鲈形体指标的影响见表14。肝体比呈现先上升后下降趋势，D80组肝体比显著高于D240，D300和和D600组，和对照组没有显著差异。6个处理组之间的肥满度、脾脏指数、脏体比均没有显著差异(P＞0.05)。The effects of adding bile acid to the feed on body index of largemouth bass are shown in Table 14. The liver-body ratio increased first and then decreased. The liver-body ratio of the D80 group was significantly higher than that of the D240, D300, and D600 groups, and there was no significant difference from the control group. There was no significant difference in fatness, spleen index, and visceral body ratio among the 6 treatment groups (P> 0.05).
表14不同剂量胆汁酸对大口黑鲈形体指标的影响(Mean±S.E.)Table 14 Effects of different doses of bile acids on body indexes of largemouth bass (Mean ± S.E.)
Figure PCTCN2019095091-appb-000016
Figure PCTCN2019095091-appb-000016
4.3饲料中添加不同剂量的胆汁酸对大口黑鲈血浆脂眆代谢指标及抗氧化功能的影响4.3 Effects of adding different doses of bile acids to the plasma lipid metabolism and antioxidant function of largemouth bass
饲料中添加胆汁酸对大口黑鲈血浆脂肪代谢指标的影响见表15。各个处理组之间TG和ALP没有显著差异(P＞0.05)。TC和HDL-C随饲料中胆汁酸的添加水平呈上升趋势，并在D240组达到最高(P＜0.05)：TBILI星现不规律显著差异(P＜0.05)，其中D0组与其余各组均差异不显著(P＞0.05)。The effects of adding bile acid to the feed on plasma lipid metabolism indexes of largemouth bass are shown in Table 15. There was no significant difference in TG and ALP between the treatment groups (P> 0.05). TC and HDL-C showed an increasing trend with the addition of bile acids in the feed, and reached the highest in the D240 group (P <0.05): TBILI star irregularities were significantly different (P <0.05), of which the D0 group and the other groups were all The difference was not significant (P> 0.05).
饲料中添加胆汁酸对大口黑鲈血浆抗氧化指标的影响见表16。由表16可以得出，MDA、GST各组均无差异(P＞-0.05)，SOD、T-AOC、GSH1-PX呈显著上升趋势(P＜0.05)。The effect of adding bile acid to the feed on the plasma antioxidant indexes of largemouth bass is shown in Table 16. It can be concluded from Table 16 that there was no difference between the MDA and GST groups (P> -0.05), and SOD, T-AOC, and GSH1-PX showed a significant upward trend (P <0.05).
表15不同剂量胆汁酸对大口黑鲈血浆脂肪代谢指标的影响(Mean±S.E)Table 15 Effect of different doses of bile acids on plasma lipid metabolism indexes of largemouth bass (Mean ± S.E)
Figure PCTCN2019095091-appb-000017
Figure PCTCN2019095091-appb-000017
Figure PCTCN2019095091-appb-000018
Figure PCTCN2019095091-appb-000018
表16不同剂量胆汁酸对大口黑鲈血浆抗氧化指标的影响(Mean±S.E.)Table 16 Effect of different doses of bile acids on plasma antioxidant indexes of largemouth bass (Mean ± S.E.)
Figure PCTCN2019095091-appb-000019
Figure PCTCN2019095091-appb-000019
4.4饲料中添加不同剂量的胆汁酸对大口黑鲈肝脏组织中抗氧化指标及胆汁酸和胆固醇含量的影响4.4 Effects of different doses of bile acids in feed on antioxidant indexes and bile acid and cholesterol content in liver tissues of largemouth bass
饲料中添加胆汁酸对大口黑鲈肝脏抗氧化指标及胆汁酸和胆固醇含量的影响见表17。由表17可得，GSH1-PX、T-AOC呈现不规律显著差异(P＜0.05)，且D0组与其余各组无显著性差异。D600组GST最低，并显著低于DO，D160和D240组(P＜0.05)。MDA呈下降趋势且D300组最低。SOD呈显著上升趋势(P＜0.05)。肝脏中TC没有显著差异，肝脏TBA随饲料中胆汁酸的添加呈现上升趋势，并在D240组达到平台期(P＞0.05)。依据折线回归模型，以大口黑鲈肝脏总胆汁酸含量为指标时，饲料中胆汁酸的最适添加量为253.8mgkg(图7)。See Table 17 for the effects of adding bile acids to the liver on antioxidant indexes and bile acids and cholesterol levels in largemouth bass. As can be seen from Table 17, GSH1-PX and T-AOC showed irregular differences (P <0.05), and there was no significant difference between the D0 group and the other groups. The GST was lowest in D600 group and significantly lower than that in DO, D160 and D240 groups (P <0.05). MDA showed a downward trend and the lowest in the D300 group. SOD showed a significant upward trend (P <0.05). There was no significant difference in TC in the liver. The liver TBA showed an increasing trend with the addition of bile acids in the feed, and reached the plateau stage in the D240 group (P> 0.05). According to the polyline regression model, when the total bile acid content in the liver of largemouth bass was used as an index, the optimal amount of bile acid in the feed was 253.8 mgkg (Figure 7).
表17不同剂量胆汁酸对大口黑鲈肝脏组织中抗氧化及胆汁酸代谢相关指标的影响(Mean±S.E.)Table 17 Effects of different doses of bile acids on antioxidant and bile acid metabolism-related indexes in largemouth bass (Mean ± S.E.)
Figure PCTCN2019095091-appb-000020
Figure PCTCN2019095091-appb-000020
Figure PCTCN2019095091-appb-000021
Figure PCTCN2019095091-appb-000021
4.5饲料中添加不同剂量的胆汁酸对大口黑鲈肠道消化酶活性及胆囊胆汁酸含量的影响4.5 Effects of adding different doses of bile acids to the intestinal digestive enzymes and gallbladder bile acid content of largemouth bass
由表18可见，大口黑鲈中肠淀粉酶含量显示不规律上升趋势，D300组达到最高(P＜0.05)，中肠脂肪酶显示不规律性差异(P＜0.05)。后肠淀粉酶在D600达到最高，显著高于其他各组；后肠脂肪呈先升后降的趋势，D80组达到最高，但各组均与对照组没有显著差异。各组胆囊胆汁酸含量呈显著上升趋势，且最高的D600组胆囊胆汁酸含量约为对照组的4.9倍。As can be seen from Table 18, the midgut amylase content of largemouth bass showed an irregular upward trend, reaching the highest in the D300 group (P <0.05), and the midgut lipase showed irregular differences (P <0.05). The hindgut amylase reached the highest at D600, which was significantly higher than other groups; the hindgut fat increased first and then decreased, and the D80 group reached the highest, but there was no significant difference between each group and the control group. The gallbladder bile acid content in each group showed a significant upward trend, and the highest gallbladder bile acid content in the D600 group was about 4.9 times that of the control group.
表18不同剂量胆汁酸对大口黑鲈肠道消化酶活性及胆囊胆汁酸含量的影响(Mean±S.E.)Table 18 Effects of different doses of bile acids on intestinal digestive enzyme activity and gallbladder bile acid content in largemouth bass (Mean ± S.E.)
Figure PCTCN2019095091-appb-000022
Figure PCTCN2019095091-appb-000022
本发明通过实验检测了以下项目：生长性能：存活率、特定生长率、增重率、饲料系数、摄食率、肥满度、肝体比、脏体比；血浆脂肪代谢及抗氧化性能：ALP、TPRO、ALB、TBILI、TC、TG、HDL、SOD、MDA、T-AOC、GST、GST-PX；以及肝脏及胆囊的胆汁酸含量。The present invention tests the following items through experiments: growth performance: survival rate, specific growth rate, weight gain rate, feed coefficient, feeding rate, fullness, liver-body ratio, viscera-body ratio; plasma fat metabolism and antioxidant performance: ALP , TPRO, ALB, TBILI, TC, TG, HDL, SOD, MDA, T-AOC, GST, GST-PX; and the bile acid content of the liver and gallbladder.
本发明通过试验结果表明：饲料中添加适量胆汁酸可有效促进摄食和生长性能，但同时增加肝脏总胆汁酸含量。分别以特定生长率、摄食率和肝脏中总胆汁酸含量为指标，通过回归模型分析获得大口黑鲈饲料中胆汁酸最适添加量分别为440.5mg/kg、283.3mg/kg和253.8mg/kg。方差分析结果显示饲料中添加300mg/kg胆汁酸，大口黑鲈获得最高生长性能，且肝脏胆汁酸含量与D240组没有差异。在以上添加范围内，胆汁酸有效降低大口黑鲈肝脏中MDA含量，提高抗氧化能力；通过提高血浆中HDL-C含量显著促进脂肪代谢，同时有效提高了肠道淀粉酶和脂肪酶活性。综合生长性能，免疫抗氧化指标及胆汁酸在肝脏中的蓄积水平建议大口黑鲈饲料中胆汁酸推荐添加量为253.8mg/kg-283.3mg/kg，最高推荐剂量为300mg/kg。The test results of the present invention show that adding a proper amount of bile acid to the feed can effectively promote feeding and growth performance, but at the same time increase the total bile acid content of the liver. Using the specific growth rate, food intake rate and total bile acid content in the liver as indicators, the optimal bile acid additions in the feed for largemouth bass were 440.5 mg / kg, 283.3 mg / kg, and 253.8 mg / kg by regression model analysis. . The analysis of variance showed that adding 300mg / kg bile acid to the feed, the maximum growth performance of largemouth bass was obtained, and the content of liver bile acid was not different from that of D240 group. Within the above range of addition, bile acid effectively reduces the MDA content in the liver of largemouth bass and improves the antioxidant capacity; by increasing the HDL-C content in the plasma, it significantly promotes fat metabolism and effectively improves intestinal amylase and lipase activities. Comprehensive growth performance, immune anti-oxidation index and bile acid accumulation level in the liver, the recommended amount of bile acid in largemouth bass feed is 253.8mg / kg-283.3mg / kg, the maximum recommended dose is 300mg / kg.
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, it will still be possible for those skilled in the art to understand the foregoing embodiments. The recorded technical solutions are modified, or some technical features are replaced equivalently; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed in the present invention.

Claims (11)

  1. 胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用。Application of bile acid in preparing additives for improving growth performance of weaned piglets.
  2. 根据权利要求1所述的胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用，其特征在于：所述胆汁酸包括以下成分：按质量比计，猪胆酸5％-20％、猪去氧胆酸55％-75％和鹅去氧胆酸10％-20％。The use of bile acid according to claim 1 in the preparation of an additive for improving the growth performance of weaned piglets, wherein the bile acid comprises the following components: in terms of mass ratio, pig bile acid is 5% -20%, Hododeoxycholic acid 55% -75% and chenodeoxycholic acid 10% -20%.
  3. 根据权利要求1所述的胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用，其特征在于：所述胆汁酸的制备方法包括如下步骤：The use of bile acid according to claim 1 in preparing an additive for improving the growth performance of weaned piglets, wherein the method for preparing the bile acid comprises the following steps:
    (1)皂化(1) Saponification
    称取粉碎的胆膏，加入反应釜中，加入氢氧化钠溶液，升温并保持沸腾状态6-24h，将皂化液冷却至固液分层，将上清液去除，固体即为胆膏皂化品；Weigh the pulverized bile, add it to the reaction kettle, add sodium hydroxide solution, heat up and keep boiling for 6-24h, cool the saponified liquid to solid-liquid layering, and remove the supernatant. The solid is the saponified bile. ;
    (2)脱色(2) Discoloration
    向胆膏皂化品中加水并加热使胆膏皂化品完全溶解，然后加入双氧水，搅拌均匀后反应，反应完毕后，将反应溶液过滤得滤液；Add water to the gall saponified product and heat to completely dissolve the gall saponified product, then add hydrogen peroxide, stir and react after the reaction is uniform. After the reaction is completed, filter the reaction solution to obtain a filtrate;
    (3)酸化(3) Acidification
    将所述滤液冷却至室温，缓慢加入盐酸溶液，并搅拌，待溶液pH值在3-4时，停止加酸，此时得到白色固形物；The filtrate was cooled to room temperature, and the hydrochloric acid solution was slowly added and stirred. When the pH of the solution was 3-4, the addition of acid was stopped, and a white solid was obtained at this time;
    (4)纯化(4) Purification
    将所述白色固形物过滤后，加水冲洗，待过滤出的水检测呈中性为水洗结束；将过滤后的白色固形物放入烘箱中干燥至水分不高于10％，得胆汁酸粗品；After filtering the white solids, rinse with water, and the water to be filtered will be neutral when the washing ends; the filtered white solids are dried in an oven to a moisture content of not more than 10%, and a crude bile acid is obtained;
    (5)重结晶(5) Recrystallization
    将得到的胆汁酸粗品投入萃取罐中，加入乙酸乙酯搅拌溶解，待胆汁酸粗品基本溶解，加入无水硫酸钠脱水，收集溶液，将溶液进行减压浓缩，将浓缩液压滤得到胆汁酸；Put the obtained crude bile acid into an extraction tank, add ethyl acetate and stir to dissolve. After the crude bile acid is basically dissolved, add anhydrous sodium sulfate to dehydrate, collect the solution, concentrate the solution under reduced pressure, and concentrate the hydraulic filtration to obtain bile acid;
    (6)干燥(6) Drying
    将得到的胆汁酸放入烘箱，通蒸汽进行真空干燥，待胆汁酸水分不超过1％，则得到所述胆汁酸。The obtained bile acid is put into an oven, and is subjected to vacuum drying through steam. When the bile acid moisture does not exceed 1%, the bile acid is obtained.
  4. 根据权利要求1所述的胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用，其特征在于：所述断奶仔猪为断奶后1-2周的仔猪。The use of bile acid according to claim 1 in the preparation of an additive for improving the growth performance of weaned piglets, wherein the weaned piglets are piglets 1-2 weeks after weaning.
  5. 根据权利要求1所述的胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用，其特征在于：所述胆汁酸添加到含有米糠油和/或全脂豆粕的日粮中。The use of bile acid according to claim 1 in preparing an additive for improving the growth performance of weaned piglets, wherein the bile acid is added to a diet containing rice bran oil and / or full fat soybean meal.
  6. 根据权利要求5所述的胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用，其特征在于：所述胆汁酸的添加量为日粮总质量的0.03％～0.1％。The use of bile acid according to claim 5 in the preparation of an additive for improving the growth performance of weaned piglets, wherein the added amount of the bile acid is 0.03% to 0.1% of the total mass of the diet.
  7. 根据权利要求5所述的胆汁酸在制备用于提高断奶仔猪生长性能的添加剂中的应用，其特征在于：所述米糠油用量占日粮总量的质量比为0.7％，断奶仔猪日增重最高且料肉比最低。The application of bile acid according to claim 5 in the preparation of an additive for improving the growth performance of weaned piglets, characterized in that the mass ratio of the amount of rice bran oil to the total diet is 0.7%, and the daily weight gain of weaned piglets The highest and lowest meat-to-meat ratio.
  8. 胆汁酸在制备用于牛蛙的饲料添加剂中的应用。Application of bile acid in preparing feed additive for bullfrog.
  9. 根据权利要求8所述的胆汁酸在制备用于牛蛙的饲料添加剂中的应用，其特征在于：所述饲料中添加100mg/kg-200mg/kg胆汁酸。The application of the bile acid according to claim 8 in the preparation of a feed additive for a bullfrog, characterized in that: 100 mg / kg-200 mg / kg of bile acid is added to the feed.
  10. 胆汁酸在制备用于大口黑鲈的饲料添加剂中的应用。Application of bile acid in the preparation of feed additives for largemouth bass.
  11. 根据权利要求10所述的胆汁酸在制备用于大口黑鲈的饲料添加剂中的应用，其特征在于：大口黑鲈饲料中胆汁酸的添加量为200mg/kg-450mg/kg。The use of bile acids in the preparation of feed additives for largemouth bass according to claim 10, characterized in that the amount of bile acids in the largemouth bass feed is 200mg / kg-450mg / kg.
PCT/CN2019/095091 2018-07-09 2019-07-08 Bile acid, preparation method thereof, and application of same in preparing animal feed additive WO2020011128A1 (en)

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CN201810747818.8A CN108522860A (en) 2018-07-09 2018-07-09 Application of the bile acid in preparing the feed addictive for bullfrog
CN201810747819.2A CN108783015A (en) 2018-07-09 2018-07-09 Application of the bile acid in preparing the additive for improving Growth Performance of Weaning Piglets
CN201810747818.8 2018-07-09
CN201810747819.2 2018-07-09
CN201810747805.0A CN108522858A (en) 2018-07-09 2018-07-09 Application of the bile acid in preparing the feed addictive for Micropterus salmoides
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