Feed composition, method for raising Chinese soft-shelled turtle and uses thereof

Abstract

The present application discloses a feed composition, a feeding method for Chinese soft-shelled turtles and uses thereof. The feed composition includes, by mass parts, white fish meal containing 65% protein, white fish meal containing 58% protein, chicken meal, soybean meal, high-gluten flour, raw cassava starch, soybean oil, calcium dihydrogen phosphate, choline chloride, table salt, a premix for turtles and soft-shelled turtles, L-lysine hydrochloride, DL-methionine and 75% tannic acid. The feed composition can promote the growth of Chinese soft-shelled turtles, improve the antioxidant capacity of Chinese soft-shelled turtles, enhance the anti-inflammatory response ability and improve the immune function of Chinese soft-shelled turtles.

Description

Technical Field

[0001] This application relates to the technical field of the breeding of Chinese soft-shelled turtles (Pelodiscus sinensis), and specifically relates to a feed composition, a breeding method and uses of Chinese soft-shelled turtles. Background Art

[0002] The Chinese soft-shelled turtle (Pelodiscus sinensis), commonly known as soft-shelled turtle, turtle, etc., belongs to the class Reptilia, order Testudinata, family Trionychidae, and genus Pelodiscus. The muscle of the Chinese soft-shelled turtle is rich in 19.63% - 21.42% protein and 16 kinds of amino acids, with delicious meat quality, having both edible and medicinal values. Due to the increasing market demand, since the late 1990s, the breeding mode of Chinese soft-shelled turtles has gradually developed from pond breeding to high-density industrialized breeding mode. According to the China Fisheries Statistical Yearbook 2023, the breeding output of Chinese soft-shelled turtles reached 373,709 tons in 2022. In the high-density industrialized breeding environment, Chinese soft-shelled turtles are extremely vulnerable to various stress factors such as malnutrition, temperature fluctuations, and pathogen infections, resulting in the death of breeding individuals and causing economic losses. At the same time, these stress factors will also reduce the muscle quality of Chinese soft-shelled turtles. Therefore, in the context of "antibiotic ban" and the development trend of green and healthy breeding, it is of great significance to find natural and safe feed additives to promote the growth performance of Chinese soft-shelled turtles and improve their stress resistance ability, and even to promote the large-scale development of the Chinese soft-shelled turtle breeding industry. Summary of the Invention

[0003] In view of this, the embodiments of this application at least disclose the following technical solutions:

[0004] In a first aspect, an embodiment discloses a feed composition. The feed composition includes, by mass parts, 3800 - 4200 parts of white fish meal containing 65% protein, 1000 - 2000 parts of white fish meal containing 58% protein, 300 - 700 parts of chicken meal, 600 - 1000 parts of soybean meal, 1800 - 2200 parts of high-gluten flour, 300 - 600 parts of raw cassava starch, 100 - 300 parts of soybean oil, 100 - 300 parts of calcium dihydrogen phosphate, 10 - 30 parts of choline chloride, 10 - 30 parts of table salt, 10 - 30 parts of a premix for turtles and soft-shelled turtles, 50 - 150 parts of L-lysine hydrochloride, 20 - 60 parts of DL-methionine, and 1 - 60 parts of 75% tannic acid.

[0005] In some embodiments, the feed composition includes, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 2043 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 7 parts of 75% tannic acid.

[0006] In some embodiments, the feed composition includes, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 2037 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 13 parts of 75% tannic acid.

[0007] In some embodiments, the feed composition includes, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 2023 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 27 parts of 75% tannic acid.

[0008] In some embodiments, the feed composition includes, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 1997 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 53 parts of 75% tannic acid.

[0009] In the second aspect, the embodiment discloses a method for raising Chinese soft-shelled turtles, which comprises: feeding Chinese soft-shelled turtles with a basic feed for temporary raising for 2 weeks; and after the temporary raising, fasting for 24 hours, and then raising the soft-shelled turtles with the feed composition described in the first aspect.

[0010] In some embodiments, the basal feed, by mass parts, comprises 3,800 - 4,200 parts of white fish meal with 65% protein, 1,000 - 2,000 parts of white fish meal with 58% protein, 300 - 700 parts of chicken meal, 600 - 1,000 parts of soybean meal, 1,800 - 2,200 parts of high-gluten flour, 300 - 600 parts of raw cassava starch, 100 - 300 parts of soybean oil, 100 - 300 parts of monocalcium phosphate, 10 - 30 parts of choline chloride, 10 - 30 parts of table salt, 10 - 30 parts of premix for turtles and soft-shelled turtles, 50 - 150 parts of L-lysine hydrochloride, and 20 - 60 parts of DL-methionine.

[0011] In some embodiments, the conditions for temporary rearing include: temporary rearing is carried out in a cleaned and disinfected polyethylene pool of 2m×1m×0.25m.

[0012] In some embodiments, the conditions for feeding include: feeding the feed composition described in the first aspect 2 times at 9:00 and 17:00 every day.

[0013] In some embodiments, the conditions for feeding include: feeding in a polyethylene pool of 1m×1m×0.25m.

[0014] In some embodiments, the conditions for feeding include: during the non-feeding period, aerating the water body of the polyethylene pool with an air pump for 6 hours every day.

[0015] In some embodiments, the conditions for feeding include: changing the water in the water body of the polyethylene pool once every 3 days, and the water change volume is 1 / 3 of the total volume.

[0016] In some embodiments, the conditions for feeding include: maintaining the water temperature of the polyethylene pool at 28±1°C, the pH at 8.0±0.4, the dissolved oxygen controlled at 6.0±1.7mg / L, NH3-N at 4.0±1.2mg / L, NO 2- The control level is 1.0±0.4mg / L, and the water body alkalinity control level is 45±4.

[0017] In the third aspect, the embodiments disclose the use of the feed composition described in the first aspect for at least one of the following: promoting the growth of Chinese soft-shelled turtles; improving the antioxidant capacity of Chinese soft-shelled turtles; enhancing the anti-inflammatory response ability; improving the immune function of Chinese soft-shelled turtles.

[0018] In this application, the term "part" refers to any weight as one "part", such as 1000kg, 100kg, 10kg, 1kg, 100g, 10g, 1g, 100mg, 10mg, 1mg or other weights, and can also refer to mass percentages, such as 100%, 50%, 10%, 1%, 0.07%, 0.13%, 0.27%, 0.53% or 0.01% as one "part".

[0019] Compared with the prior art, the present application has at least the following technical effects:

[0020] In the present application, a feed composition is obtained by adding tannic acid to a basic feed. It is found that tannic acid has an impact on the growth performance, physiological metabolism indexes and muscle amino acid composition of Chinese soft-shelled turtles, providing a theoretical basis for the use of tannic acid in the feeding production of Chinese soft-shelled turtles, and at the same time being able to provide a new functional feed additive for the healthy feeding industry of Chinese soft-shelled turtles, with a view to improving the production performance of factory farming of Chinese soft-shelled turtles. Description of the Drawings

[0021] Figure 1 Graph showing the effect of feed compositions containing different tannic acids provided in the examples on the activity of plasma antioxidant indexes of Chinese soft-shelled turtles.

[0022] Figure 2 Graph showing the effect of feed compositions containing different tannic acids provided in the examples on the activity of plasma stress indexes of Chinese soft-shelled turtles.

[0023] Figure 3 Graph showing the effect of feed compositions containing different tannic acids provided in the examples on the concentration of plasma metabolic wastes of Chinese soft-shelled turtles.

[0024] Figure 4 Graph showing the effect of feed compositions containing different tannic acids provided in the examples on the relative mRNA expression levels of antioxidant-related genes in the liver of Chinese soft-shelled turtles. Detailed Description of the Embodiments

[0025] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not described in detail and separately in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail and particularly are all conventional feeding methods and can be known from the prior art.

[0026] To study the effects of tannic acid on the growth performance, physiological metabolism indexes and muscle amino acid composition of Chinese soft-shelled turtles, the embodiments provide a feed composition, a method for feeding using the feed composition, and the use of the feed composition in Chinese soft-shelled turtles.

[0027] 1. Feed Composition

[0028] As shown in Table 1, in the examples, tannic acid was added at dosages of 0.05% (0.05TA), 0.1% (0.1TA), 0.2% (0.2TA), and 0.4% (0.4TA) to replace an equal mass of high-gluten flour based on the basic feed for Chinese soft-shelled turtles, and four feed compositions were prepared. Among them, the tannic acid used was purchased from Guangdong Pulan Biotechnology Co., Ltd., with a purity of 75%. All feed raw materials were pulverized by an ultrafine pulverizer and then passed through an 80-mesh sieve. After weighing, the raw materials were fully mixed by a mixer, granulated and puffed into 1.5-mm puffed feed by an extruder, dried at 55°C and then cooled to room temperature, and packed into sealed bags for storage and use at room temperature. The feed raw materials and processing equipment were kindly provided by (Guangdong Linkun Group Co., Ltd.).

[0029] Table 1 Composition of feed composition (mass percentage%)

[0030]

[0031]

[0032] 2. Feeding method for Chinese soft-shelled turtles

[0033] On the one hand, the examples disclose a feeding method for Chinese soft-shelled turtles. The feeding method includes: feeding the Chinese soft-shelled turtles with the basic feed for 2 weeks for temporary rearing; and after the temporary rearing ends and fasting for 24 h, feeding them with the feed composition described in the first aspect.

[0034] (1) Specific feeding process

[0035] The Chinese soft-shelled turtles used for feeding were obtained from Guangdong Caixing Industrial Co., Ltd. and were fed in the indoor feeding system of the turtle and soft-shelled turtle breeding base of the Pearl River Fisheries Research Institute in Guangzhou. Before the feeding started, the Chinese soft-shelled turtles were temporarily raised in a polyethylene pond (2m×1m×0.25m) that had been cleaned and disinfected for 2 weeks. During the temporary raising period, a basal diet without tannic acid was fed. After the temporary raising ended, they were fasted for 24h, and 1050 individuals with the same specifications, good condition, and sufficient vitality were randomly selected as the turtles for feeding. The 1050 Chinese soft-shelled turtles (with an average weight of 12.54±0.54g) were randomly assigned to 5 feeding groups, with 3 replicates in each group and 70 individuals in each replicate. Each replicate group was fed in a 1m×1m×0.25m polyethylene pond, and the 5 feeding groups were fed diets with tannic acid contents of 0 (Group C, as the control group), 0.05% (0.05TA), 0.1% (0.1TA), 0.2% (0.2TA), and 0.4% (0.4TA), respectively. They were fed regularly twice a day (at 09:00 and 17:00), and were fed to satiety each time until there was a small amount of feed settling in the pond and most of the Chinese soft-shelled turtles had no feeding behavior. 1h after the feeding ended, the residual bait and feces were immediately sucked out and collected. The feeding period was 60 days. During the non-feeding period, the water body was aerated with an air pump for 6 hours every day; the water was changed once every 3 days, and the amount of water changed was 1 / 3 of the total volume; the water temperature was maintained at 28±1℃ with an electric heater, the pH of the water body was 8.0±0.4, the dissolved oxygen was 6.0±1.7mg / L, NH3-N was 4.0±1.2mg / L, NO 2- was 1.0±0.4mg / L, and the alkalinity of the water body was 45±4. During the feeding period, the amount of feed consumed by the Chinese soft-shelled turtles in each pond and the death situation of the fed individuals were recorded every day.

[0036] (2) Sample collection

[0037] On the 0th, 30th, and 60th days after the feeding of the diet, all the fed individuals in the feeding groups were weighed and 9 individuals from each group were sampled. Before weighing and sampling, they were fasted for 24h. 9 Chinese soft-shelled turtles were randomly selected from each replicate. After drying the surface moisture of the body, they were quickly anesthetized with 1000mg / L of MS-222, and then decapitated to collect blood. The blood was dropped into an anticoagulation tube containing sodium heparin. The collected blood was placed at 4℃ and allowed to stand for 4h, and then centrifuged at 4℃ and 3000r / min for 15min in a refrigerated centrifuge. Subsequently, the supernatant was immediately taken and placed in a 2mL sterile centrifuge tube and stored in a -80℃ refrigerator for the detection of plasma biochemical indexes. After blood sampling, the Chinese soft-shelled turtles were dissected, and the liver tissue was stored in liquid nitrogen for subsequent detection of gene mRNA expression levels.

[0038] (3) Calculation of growth performance indexes

[0039] The calculation formulas for the growth performance-related indexes are as follows:

[0040] Weight gain rate (WGR, %) = 100% × (W t - W0) / W0;

[0041] Specific growth rate (SGR, %) = 100% × [lnW t - lnW0 / t];

[0042] Feeding rate (FR, %) = 100% × F / [t × (W0 + W t ) / 2];

[0043] Feed conversion ratio (FCR, %) = 100% × F / (W t - W0);

[0044] Protein efficiency ratio (PER, %) = 100% × (W t - W0) / (F × CP).

[0045] In the formulas, W0 represents the initial weight (g) of the Chinese soft-shelled turtle, W t represents the final weight (g) of the Chinese soft-shelled turtle, t represents the number of feeding days, F represents the dry matter weight (g) of the fed feed, and CP represents the crude protein content (%) in the feed

[0046] (4) Plasma biochemical indexes

[0047] The activities or contents of the plasma biochemical indexes of the Chinese soft-shelled turtle were detected using kits from Nanjing Jiancheng Bioengineering Institute. The detection indexes and kit product numbers are as follows: total superoxide dismutase T-SOD (A001-1-2), catalase CAT (A007-1-1), glutathione peroxidase GSH-Px (A005-1-2), aspartate aminotransferase ALT (C009-2-1), alanine aminotransferase AST (C010-2-1), alkaline phosphatase AKP (A059-2-2), total bilirubin TBIL (C019-1-1), and urea UREA (M004-1-1).

[0048] (5) Detection of muscle amino acid content

[0049] The dissected Chinese soft-shelled turtle was placed in a high-temperature sterilizer, taken out after steaming at high temperature for 15 min, and the muscle tissues of the limbs and neck were carefully peeled off. The peeled tissues were placed in an oven at 65 °C, dried until the weight no longer decreased, taken out, mashed and mixed with a high-speed tissue homogenizer for detecting the muscle amino acid composition. The detection of the muscle amino acid content of the Chinese soft-shelled turtle after 60 days of feeding was carried out with reference to the national standard GB5009.124—2016, and determined by the hydrolysis amino acid method using an LA-8080 automatic amino acid analyzer (Japan).

[0050] (6) RT-PCR

[0051] The total RNA of Chinese soft-shelled turtle liver tissue was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China). 1 μL of the RNA sample was electrophoresed in 1% agarose gel to detect RNA integrity, and the RNA concentration was measured using a NanoDrop One / OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher, Waltham, USA). 1 μg of cDNA was synthesized using the HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, Nanjing, China).

[0052] Using Applied QuantStudio TM 6 Flex (ABI, Waltham, USA) real-time fluorescence quantitative detection system was used for qPCR analysis, and the fluorescence quantitative reagent was iTaq Universal SYBR Green Supermix (Bio-Rad, California, USA). Primers for superoxide dismutase 1 (Sod1), superoxide dismutase 2 (Sod2), catalase (Cat), glutathione peroxidase 3 (Gpx3), and glutathione peroxidase 4 (Gpx4) genes were designed according to the gene sequences in the NCBI database. Chinese soft-shelled turtle Efα1 was selected as the internal reference gene, and Primer Premier 6.0 software was used for primer design. The primer sequences are shown in Table 2. qPCR reaction system: 2×SYBR Green Supermix 10 μL, 1 μL each of 10 μM forward and reverse primers, 200 ng of cDNA sample, and made up to 20 μL with ultrapure water. qPCR reaction conditions: 95 °C, 5 min; (95 °C, 5 s; 60 °C, 30 s), 40 cycles. In the qPCR experiment, 6 biological replicates were measured for each treatment group, and 3 technical replicates were set for each replicate. The experimental results were referenced to the control group, and the 2 -△△Ct method was used to calculate the expression fold change of each gene in the treatment group relative to the control group.

[0053] Table 2 Primer sequences for fluorescence quantitative PCR

[0054]

[0055] (6) Data statistics and analysis

[0056] The feeding data were statistically analyzed using SPSS 29.0 software. Among them, the indicators related to growth performance were processed by two-way ANOVA (Two-Way ANOVA), and the plasma biochemical indicators, liver gene expression levels, and muscle amino acid contents were all processed by one-way ANOVA (One-Way ANOVA). The Turkey test was used for post hoc multiple comparison analysis of the differences between groups, and the results were expressed as "mean ± standard deviation". P<0.05 represented significant differences, and P<0.01 represented extremely significant differences.

[0057] 3. Use of the feed composition in promoting the growth performance of Chinese soft-shelled turtles

[0058] The examples disclose the use of the above feed composition in promoting the growth performance of Chinese soft-shelled turtles.

[0059] Table 3 shows that the feed compositions provided in each example were fed to Chinese soft-shelled turtles for 60 days, and it was found that the feed compositions provided in the examples of the present application could promote their growth performance compared with the basal diet. In Table 3, FBW, WGR, SGR, FR, FCR, and PER represent final body weight (g), weight gain rate (%), specific growth rate (%), feed intake rate (%), feed conversion rate (%), and protein efficiency ratio (%) in turn. In Table 3, different letters with superscripts in the same column of data indicate significant differences in the same index among different groups at the same feeding time (P<0.05). The * with superscript in the same column of data indicates significant differences in the same index between different feeding times in the same group (P<0.05). No characters indicate no significant differences (P>0.05). The ** with superscript in the two-way ANOVA indicates extremely significant effects (P<0.01).

[0060] After 60 days of feeding, FBW, WGR, and SGR in all tannic acid feeding groups were significantly higher than those in the control group (P<0.05), and WGR and SGR in the 0.2% and 0.4% tannic acid groups were significantly higher than those in the 0.05% and 0.1% tannic acid groups (P<0.05). The SGR in the 0.1% tannic acid group at 60 days was significantly higher than that at 30 days (P<0.05). After 30 days of feeding, FCR and PER in the 0.1% tannic acid group were significantly higher than those in the control group (P<0.05). After 60 days of feeding, FR in the 0.05% and 0.1% tannic acid groups, FCR in the 0.2% and 0.4% tannic acid groups, and PER in the 0.1%-0.4% tannic acid groups were all significantly higher than those in the control group (P<0.05). Two-way ANOVA showed that there were significant interaction effects of tannic acid addition amount and feeding days on FBW, WGR, SGR, FCR, and PER of Chinese soft-shelled turtles (P<0.05).

[0061] Table 3 Effects of tannic acid on the growth performance of Chinese soft-shelled turtles

[0062]

[0063] This indicates that after feeding the soft-shelled turtle with the feed composition containing 0.05%-0.4% tannic acid provided in the examples for 60 days, the final weight, feeding rate, weight gain rate, specific growth rate and feed conversion rate of the soft-shelled turtle can be significantly improved, especially the feed compositions containing 0.2% and 0.4% tannic acid.

[0064] 4. Use of the feed composition in improving the antioxidant capacity of soft-shelled turtles

[0065] As Figure 1 shown, after 30 days of feeding, the T-SOD activity in the 0.1% tannic acid group was significantly higher than that in the control group and the 0.05% tannic acid group (P<0.05); there was no significant difference in GSH-Px among groups (P>0.05); the CAT activities in the 0.1%, 0.2% and 0.4% tannic acid groups were significantly lower than those in the control group (P<0.05). After 60 days of feeding, compared with the control group, the T-SOD activities in the 0.2% and 0.4% tannic acid groups were significantly decreased (P<0.05); the GSH-Px activities in the 0.05%, 0.1% and 0.4% tannic acid groups were significantly decreased (P<0.05); the CAT activities in the 0.2% and 0.4% tannic acid groups were significantly lower than those in the control group and the 0.05% tannic acid group (P<0.05).

[0066] As Figure 4 shown, after 60 days of feeding, the mRNA levels of Sod1, Sod2 and Cat in the liver of the 0.2% tannic acid group were significantly higher than those in the control group (P<0.05), and there was no significant difference in the remaining tannic acid groups compared with the control group (P>0.05). The mRNA expression levels of Gpx3 in the 0.05% and 0.2% tannic acid groups were significantly higher than those in the control group (P<0.05). The mRNA expression levels of Gpx4 in the 0.1% and 0.2% tannic acid groups were higher than those in the control group and the other tannic acid dosage groups (P<0.05).

[0067] Reactive oxygen species (ROS) are highly reactive oxygen-containing derivatives produced by the aerobic metabolism of the body. When the body is stimulated by adverse factors, the production of ROS in the body will increase. Excessive ROS will attack the phospholipids on the cytoplasmic membrane and the polyunsaturated fatty acids on the membrane receptors to form lipid peroxides, causing damage to the structures of biological macromolecules such as DNA, and even leading to diseases and death of the body. Under normal conditions, the body can timely remove excessive ROS through the antioxidant defense system to maintain the balance of ROS in the body. Antioxidant enzymes are important members of the antioxidant system, mainly including SOD, GSH-Px and CAT. These antioxidant enzymes can protect cells from the damage of excessive ROS, so their activities are often used to indicate the antioxidant capacity of the body. SOD mainly includes SOD1 (Cu / ZnSOD) distributed in the cytoplasm and SOD2 (Mn / FeSOD) in the mitochondria. SOD catalyzes the dismutation reaction of superoxide to generate oxygen and hydrogen peroxide, thereby reducing the reactive oxygen radicals in the cell. GSH-Px can catalyze the reduction reaction of peroxides such as hydrogen peroxide and peroxynitrite to reduce oxidative stress damage. CAT promotes the decomposition of hydrogen peroxide into water and oxygen to remove excessive ROS.

[0068] This application found that the feed composition added with tannic acid can increase the mRNA levels of Sod1, Sod2, Cat, Gpx3 and Gpx4 in the liver of Chinese soft-shelled turtles and reduce the activities of plasma T-SOD, GSH-Px and CAT. This application found that feeding with the feed composition added with tannic acid can enhance the antioxidant capacity of the Chinese soft-shelled turtle body and strengthen the ability to scavenge ROS. This may be related to tannic acid activating the signal pathway related to antioxidants and then enhancing the expression of antioxidant enzyme genes. At the same time, the ortho-phenolic hydroxyl structure of tannic acid can combine with free radicals in the body by providing hydrogen ions, thereby blocking the free radical chain reaction, reducing the content of free radicals and improving the antioxidant capacity of the body. ALT and AST are important indicators for indicating the damage status of animal tissues.

[0069] Therefore, the embodiment discloses the use of the above feed composition in improving the antioxidant capacity of Chinese soft-shelled turtles.

[0070] 5. Use of the feed composition in improving the immune function of Chinese soft-shelled turtles

[0071] Such as Figure 2, after 30 days of feeding, there were no significant differences in the activities of plasma ALT, AST, and ALP in each tannic acid group compared with the control group (P>0.05). After 60 days of feeding, there were no significant differences in ALT and AST in all tannic acid groups compared with the control group (P>0.05), but the AST activity in the 0.4% tannic acid group was significantly higher than that in the 0.2% group (P<0.05); the ALP activity in the 0.4% tannic acid group was significantly higher than that in the control group (P<0.05), and there were no significant differences in the other groups compared with the control group (P>0.05).

[0072] Under normal conditions, the contents of ALT and AST in the blood are low. When liver damage occurs, ALT in hepatocytes enters the blood circulation, and the activity of ALT in the blood increases; when internal organs such as the heart and liver are damaged, AST in their tissues is released into the blood, resulting in an increase in the activity of AST in the blood. ALP is an important regulatory enzyme that can directly participate in the transfer and metabolism of phosphate groups and plays an important role in the immune response and material metabolism in fish. It was found in this application that the feed composition added with 0.4% tannic acid can increase the ALP activity of Chinese soft-shelled turtles. This indicates that the addition of tannic acid does not damage the internal organs such as the heart and liver of Chinese soft-shelled turtles, and at the same time, the addition of tannic acid may improve the immune function of Chinese soft-shelled turtles.

[0073] Therefore, the embodiment discloses the use of the above feed composition in improving the immune function of Chinese soft-shelled turtles.

[0074] 6. The addition of tannic acid has no adverse effects on the hemoglobin metabolism and kidney protein metabolism of Chinese soft-shelled turtles

[0075] As Figure 3 It can be seen that: when feeding for 30 days, compared with the control group, the TBIL content in the 0.05% tannic acid group was significantly increased (P<0.05), and there were no significant differences among the other groups (P>0.05); the UREA content in the 0.4% tannic acid group was significantly decreased (P<0.05). After 60 days of feeding, the TBIL contents in the 0.05% and 0.2% tannic acid groups were significantly lower than that in the control group (P<0.05); there were no significant differences in the UREA contents of each group (P>0.05).

[0076] TBIL is one of the important components of bile, mainly produced by the decomposition of hemoglobin. When liver function is abnormal, TBIL cannot be normally metabolized and accumulates in the blood, causing liver lesions. UREA is one of the final products of protein and amino acid metabolism, and its content can be used to evaluate the functional state of kidney protein metabolism. Reduced or lost kidney function will lead to the accumulation of UREA in the blood, thus causing hyperuricemia. In this application, 0.2% tannic acid significantly reduced the TBIL content of Chinese soft-shelled turtles, and 0.05%-0.4% tannic acid reduced the UREA content in the plasma of Chinese soft-shelled turtles after 30 days of feeding, indicating that the addition of tannic acid has no adverse effects on the hemoglobin metabolism and kidney protein metabolism of Chinese soft-shelled turtles.

[0077] 7. The addition of tannic acid does not affect the amino acid composition and content of soft-shelled turtle muscle

[0078] Table 4 shows that after 60 days of feeding, a total of 16 amino acids were detected in the muscle of soft-shelled turtles in each group (Table 4), including 7 essential amino acids (threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine) and 9 non-essential amino acids (aspartic acid, serine, glutamic acid, glycine, alanine, tyrosine, histidine, arginine, and proline). In Table 4, * indicates umami amino acids; # indicates essential amino acids; amino acids without symbols are non-essential amino acids. Different letters with superscripts in the same row of data indicate significant differences in the same index between different groups (P<0.05).

[0079] Table 4 Amino acid composition and content of soft-shelled turtle muscle after 60 days of feeding (wet weight g / 100g)

[0080] Amino acid Control group C 0.05TA 0.1TA 0.2TA 0.4TA Aspartic acid Asp* 7.44±0.24 7.05±0.61 7.08±0.75 6.74±0.46 6.90±0.63 Threonine Thr# 3.66±0.26 3.47±0.17 3.50±0.17 3.31±0.12 3.40±0.17 Serine Ser <![CDATA[3.33±0.10 a > <![CDATA[3.04±0.09 ab > <![CDATA[3.13±0.07 ab > <![CDATA[2.83±0.10 b > <![CDATA[3.05±0.09 ab > Glutamic acid Glu* 11.97±0.66 11.18±0.19 11.23±0.22 10.64±0.36 10.94±0.53 Glycine Gly* 5.75±0.42 4.86±0.31 5.37±0.13 4.81±0.32 5.32±0.15 Alanine Ala* 4.98±0.12 4.59±0.12 4.73±0.07 4.45±0.14 4.63±0.14 Valine Val# 3.80±0.07 3.66±0.12 3.68±0.12 3.52±0.06 3.59±0.06 Methionine Met# 2.10±0.02 2.05±0.04 2.02±0.06 1.93±0.05 1.97±0.04 Isoleucine Ile# 3.67±0.12 3.54±0.09 3.49±0.10 3.37±0.09 3.43±0.08 Leucine Leu# <![CDATA[6.17±0.17 a > <![CDATA[5.89±0.12 ab > <![CDATA[5.86±0.10 ab > <![CDATA[5.62±0.08 b > <![CDATA[5.73±0.06 b > Tyrosine Tyr 2.61±0.09 2.48±0.10 2.45±0.06 2.38±0.06 2.44±0.09 Phenylalanine Phe# 3.23±0.08 3.06±0.07 3.07±0.07 2.93±0.06 3.01±0.06 Histidine His 2.10±0.05 2.03±0.03 2.01±0.04 1.93±0.05 1.97±0.07 Lysine Lys# 6.76±0.38 6.51±0.29 6.46±0.23 6.18±0.23 6.27±0.28 Arginine Arg 5.46±0.38 5.04±0.17 5.16±0.29 4.85±0.13 5.06±0.10 Proline Pro <![CDATA[3.89±0.08 a > <![CDATA[3.40±0.06 b > <![CDATA[3.67±0.09 ab > <![CDATA[3.38±0.06 b > <![CDATA[3.61±0.08 ab > Total essential amino acids EAA 29.39±1.90 28.18±0.56 28.08±0.67 26.86±0.38 27.40±1.31 Total non-essential amino acids NEAA 47.53±2.40 43.67±2.89 44.83±2.76 42.01±2.93 43.92±3.25 Total essential amino acids / Total non-essential amino acids 0.63±0.03 0.63±0.01 0.63±0.01 0.63±0.01 0.63±0.01 Total umami amino acids UAA 30.14±2.50 27.68±2.13 28.41±2.02 26.64±2.24 27.79±2.52 Total amino acids TAA 76.92±2.48 71.85±1.99 72.91±1.98 68.87±1.91 71.32±2.63

[0081] Among the 5 groups of muscle tissues, the content of glutamic acid was the highest (10.94 - 11.97 g / 100g), followed by aspartic acid (6.74 - 7.44 g / 100g), and the content of histidine was the lowest (1.93 - 2.10 g / 100g). The serine, leucine, and proline contents in the 0.2% tannic acid group were significantly lower than those in the control group (P<0.05), and the contents of the other 16 amino acids in the tannic acid groups showed no significant changes compared with the control group (P>0.05). There were no significant differences in the total essential amino acid content, total non-essential amino acid content, and total essential amino acid / total non-essential amino acid ratio among the groups (P>0.05); the total amino acid sum in the control group was the highest, at 76.92 ± 2.48 g; the 0.2% tannic acid group was the lowest, at 68.87 ± 1.91 g; and there was no significant difference in the total amino acid sum of each tannic acid group compared with the control group (P>0.05). In addition, 4 umami amino acids were detected in the muscle: aspartic acid, glutamic acid, glycine, and alanine. The total umami amino acid content in the control group was the highest, at 30.14 g; the 0.2% tannic acid group had the lowest content, at 26.64 g, but there was no significant difference among the groups (P>0.05).

[0082] The quality of animal muscle is closely related to the composition and content of its own nutritional components. Amino acids, as the basic building blocks of proteins, their composition, types, and proportions together determine the nutritional value and different flavors of muscle. Free amino acids are the main flavoring substances and flavor precursor substances of the non-volatile taste of fish. According to the taste characteristics of amino acids, they are divided into different taste characteristic amino acids such as sweet, umami, and bitter

[46] The umami amino acids in fish muscle include aspartic acid, glutamic acid, glycine and alanine. These four amino acids were detected in the soft-shelled turtle muscle in this application, which is also consistent with the analysis results of previous scholars on the muscle composition of soft-shelled turtle. According to the ideal amino acid pattern of FAO / WHO, the ratio of essential amino acids to non-essential amino acids in the amino acid composition of better-quality proteins is above 60%. In this application, the contents of serine and leucine in the 0.2% tannic acid group were significantly reduced, but there were no significant differences in the contents of total umami amino acids, total essential amino acids, total non-essential amino acids and total amino acids. The EAA / NEAA ratios among groups were 63.1% - 63.3% and there were no differences, indicating that the addition of tannic acid does not affect the amino acid composition and content of soft-shelled turtle muscle.

[0083] As described above, only the preferred specific embodiments of this application are provided, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be covered by the protection scope of this application.

Claims

1. A feed composition for Chinese soft-shelled turtle, comprising, by mass, 3800-4200 parts of white fish meal containing 65% protein, 1000-2000 parts of white fish meal containing 58% protein, 300-700 parts of chicken meal, 600-1000 parts of soybean meal, 1800-2200 parts of high-gluten flour, 300-600 parts of raw cassava starch, 100-300 parts of soybean oil, 100-300 parts of monocalcium phosphate, 10-30 parts of choline chloride, 10-30 parts of salt, 10-30 parts of turtle and tortoise premix, 50-150 parts of L-lysine hydrochloride, 20-60 parts of DL-methionine, and 1-60 parts of tannic acid with a purity of 75%.

2. The feed composition for Chinese soft-shelled turtle according to claim 1 comprises, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 2043 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 7 parts of tannic acid with a purity of 75%.

3. The feed composition for Chinese soft-shelled turtle according to claim 1 comprises, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 2037 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 13 parts of tannic acid with a purity of 75%.

4. The feed composition for Chinese soft-shelled turtle according to claim 1 comprises, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 2023 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 27 parts of tannic acid with a purity of 75%.

5. The feed composition for Chinese soft-shelled turtle according to claim 1 comprises, by mass, 4000 parts of white fish meal containing 65% protein, 1500 parts of white fish meal containing 58% protein, 500 parts of chicken meal, 900 parts of soybean meal, 1997 parts of high-gluten flour, 500 parts of raw cassava starch, 200 parts of soybean oil, 150 parts of monocalcium phosphate, 20 parts of choline chloride, 20 parts of salt, 20 parts of turtle premix, 100 parts of L-lysine hydrochloride, 40 parts of DL-methionine, and 53 parts of tannic acid with a purity of 75%.

6. Use of the feed composition according to any one of claims 1 to 5 in promoting the growth of Chinese soft-shelled turtle.

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