Study on cholesterol replacement in Chinese mitten crab feed and method for promoting gonadal development

Abstract

The present invention discloses a method for studying cholesterol replacement in Chinese mitten crab feed and promoting gonadal development. By measuring the growth performance of Chinese mitten crabs in different cholesterol content groups and different squalene content groups and the amount of cholesterol deposition in the hepatopancreas of juvenile Chinese mitten crabs fed with different diets, a linear model of the amount of cholesterol deposition in the hepatopancreas of juvenile Chinese mitten crabs fed with different cholesterol levels was prepared, and the biological efficacy of different amounts of squalene added to replace cholesterol was analyzed. This method can be used as an effective method for screening cholesterol substitutes. In addition, the test results show that, compared with the existing commercially available Chinese mitten crab feed and chilled fish farming, by adding 0.4% squalene to the basic formula of Chinese mitten crab feed, the poor growth and gonadal development of Chinese mitten crabs in the rearing period can be effectively improved, basically achieving the effect of chilled fish farming.

Description

Technical Field

[0001] The present invention relates to a method for studying cholesterol replacement in Chinese mitten crab feed and promoting gonadal development, specifically a method for promoting the growth and gonadal development of Chinese mitten crab during the growing period and a method for studying squalene as a cholesterol substitute, belonging to the technical field of aquatic animal nutrition and feed, and aquatic animal nutrition and quality control. Background Art

[0002] The Chinese mitten crab, commonly known as river crab or hairy crab, is an important specialty economic aquaculture species in my country. Its unique flavor, rich nutrition, and delicious taste make it a favorite among consumers. Statistics from the 2023 China Fisheries Yearbook show that my country's production of Chinese mitten crab reached 815,318 tons in 2022.

[0003] In practice, artificial feeds fail to meet production requirements during the Chinese mitten crab (ERC) rearing period, especially during the roe-enriching stage. In the final rearing stage, ERCs raised on feeds experience poor growth and gonadal development, resulting in later market release and a lower proportion of high-quality crabs compared to traditional aquaculture methods like icefish. Furthermore, the gonads, as the most edible part of the Chinese mitten crab, are highly sought after by consumers. Therefore, the crab's final size and gonadal maturity directly impact its market value, leading farmers to continue using traditional aquaculture methods like icefish for economic efficiency. Due to issues like uncontrollable icefish sourcing and cumbersome feeding, technical research and development for the rearing stage of Chinese mitten crab remains a bottleneck in the industry. This, on the one hand, leads to frequent diseases, environmental pollution, and resource waste; on the other, it also poses a significant obstacle to the upgrading of the Chinese mitten crab industry to smart feeding and green production. Therefore, to meet the enormous consumer demand for Chinese mitten crab, it is necessary to develop a feed that promotes the growth and gonadal development of Chinese mitten crabs during the rearing period, replacing fresh icefish and truly implementing the use of formulated feeds throughout the entire Chinese mitten crab aquaculture process. This is an urgent issue that must be addressed to achieve sustainable development in the Chinese mitten crab aquaculture industry.

[0004] To address these issues, previous studies in our laboratory, such as Guo H, Jiang G, Dai Y, et al. Effect of dietary cholesterol on growth performance, cholesterol deposition, and lipid metabolism in adult Chinese mitten crab (Eriocheir sinensis) [J]. Aquaculture Nutrition, 2022, 2022: 2012958 and Guo H, Wang M, Wang X, et al. Effect of dietary cholesterol on ovarian development of Chinese mitten crabs (Eriocheir sinensis) [J]. Frontiers in Marine Science, 2022, 9: 1070829, have found that cholesterol may be a key factor affecting gonadal development and the application of compound feed in Chinese mitten crabs. Studies have shown that crustaceans cannot synthesize cholesterol, possibly due to a lack of the enzyme that synthesizes mevalonate into squalene. For example, Teshima Shinichi, Kanazawa Akio, Okamoto Haruto. Sterol biosynthesis from acetate and the fate of dietary cholesterol and desmosterol in crabs[J]. Journal of the Japan Society of Fisheries, 1976, 42(11): 1273-1280., cholesterol needs to be added to the feed to meet its growth and development. Related studies have shown that adding cholesterol or its substitutes to the feed can promote the gonadal development of Chinese mitten crabs, basically achieving the effect of fresh-frozen fish, such as Guo H, Wang M, Wang X, et al. Effect of dietary cholesterol on ovarian development of Chinese mitten crabs(Eriocheirsinensis)[J]. Frontiers in Marine Science, 2022, 9: 1070829. However, commercially available cholesterol mainly comes from lanolin extraction, which has a complex process and is expensive. Determining the cholesterol requirement of Chinese mitten crabs and screening its substitutes are bottleneck problems that the industry needs to solve urgently. In the past three years, our laboratory has explored the dietary cholesterol requirement of Chinese mitten crab, which is 0.2% to 0.4%, and has conducted in-depth research on the reasons and mechanisms why Chinese mitten crab cannot synthesize cholesterol, and has actively explored and screened cholesterol substitutes.

[0005] Squalene is a precursor substance for cholesterol synthesis in vertebrates and may increase the body's own cholesterol synthesis when added to feed. Moreover, with the improvement of oil refining technology, squalene, as a plant oil extract, is cheap and widely available, and has broad market application prospects. In the case of limited ability of the body to synthesize cholesterol itself, it is still unclear whether squalene can promote cholesterol synthesis, growth and gonadal development in Chinese mitten crabs. Therefore, it is necessary to evaluate the effects of squalene on the growth and gonadal development of Chinese mitten crabs during the growing period and the feasibility of squalene as a cholesterol substitute. The research and development of the present invention helps to solve the problems of growth stunting and poor gonadal development of Chinese mitten crabs due to cholesterol deficiency during the growing period, and is of great significance for promoting the industrialization of Chinese mitten crab farming. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a method for promoting the growth and gonad development of Chinese mitten crabs during the growing period and a research method for using squalene as a cholesterol substitute.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a research method for cholesterol replacement in Chinese mitten crab feed, the experimental design is as follows:

[0008] The experiment was divided into seven groups:

[0009] (1) Control group: fed with basic feed;

[0010] (2) Experimental group 1: fed with a basal diet containing 0.2% cholesterol;

[0011] (3) Experimental group 2: fed with a basal diet containing 0.4% cholesterol;

[0012] (4) Experimental group 3: fed with a basic feed containing 0.05% squalene;

[0013] (5) Experimental group 4: fed with a basic feed containing 0.1% squalene;

[0014] (6) Experimental group 5: fed with a basic feed containing 0.2% squalene;

[0015] (7) Experimental group 6: fed with a basal feed containing 0.4% squalene;

[0016] The experimental crabs were trained for 2 weeks and fed with commercially available Chinese mitten crab feed. After acclimation, 420 strong, well-proportioned male Chinese mitten crabs with an initial weight of 5.6±0.05g were selected. Each group had four replicates, with 15 crabs in each replicate. They were fed artificially to satiation and fed once every evening at 18:00 for a total of 8 weeks.

[0017] After the breeding experiment, the growth performance of Chinese mitten crabs in different cholesterol content groups and different squalene content groups, as well as the hepatopancreatic cholesterol deposition of juvenile Chinese mitten crabs fed with different diets were measured respectively. A linear model of the hepatopancreatic cholesterol deposition of juvenile Chinese mitten crabs fed with different cholesterol levels was constructed, and the biological efficacy of different amounts of squalene added to replace cholesterol was analyzed.

[0018] Preferably, the test subjects are juvenile Chinese mitten crabs, which are purchased from a local farm at the Pukou Aquatic Teaching and Research Base of Nanjing Agricultural University. During the breeding experiment, 1 / 3 new water is injected every day to maintain the water temperature at 23-29°C, the dissolved oxygen is greater than 5 mg / L, and the pH is between 7.3-8.4.

[0019] Preferably, the commercially available Chinese mitten crab feed has a protein content of 360 g / kg and a lipid content of 80 g / kg; the basal diet is prepared with fish meal, soybean meal, cottonseed meal, rapeseed meal and peanut meal as protein sources, soybean oil and fish oil as fat sources, and α-starch as a sugar source, and the basal diet is made into particles with a diameter of 2.5 mm using a meat grinder, ventilated and dried, and stored at -20°C.

[0020] Preferably, the steps for preparing the linear model of the hepatopancreatic cholesterol deposition of juvenile Chinese mitten crabs fed with different cholesterol levels are as follows:

[0021] S1. A linear model was developed between the cholesterol content of the feed and the amount of cholesterol deposited in the hepatopancreas of Chinese mitten crabs by feeding them feeds with cholesterol contents of 0%, 0.2%, and 0.4%.

[0022] S2. The correlation between squalene content in the feed and cholesterol deposition in the hepatopancreas of Chinese mitten crabs was determined by feeding them feeds containing 0.05%, 0.1%, 0.2%, and 0.4% squalene.

[0023] S3. Using the linear model established in S1, the biological efficacy of different amounts of squalene in replacing cholesterol was calculated to be 41% to 108%.

[0024] Preferably, the optimal addition amount of squalene in feed is in the range of 0.2% to 0.4%.

[0025] The present invention also discloses a method for promoting gonadal development of Chinese mitten crab, comprising adding 0.4% squalene to the basic diet of Chinese mitten crab, and the experimental design is as follows:

[0026] The trial was divided into three groups:

[0027] (1) Control group: fed with commercially available Chinese mitten crab feed;

[0028] (2) Experimental group 1: fed with fresh ice fish;

[0029] (3) Experimental group 2: fed with a basal diet supplemented with 0.4% squalene;

[0030] The experiment selected Chinese mitten crabs in the rearing stage. The crabs were trained for 2 weeks and fed a commercially available Chinese mitten crab feed. After acclimation, 120 strong, well-proportioned female Chinese mitten crabs with an initial weight of 70.37±0.47g were selected. Each group had four replicates, with 10 crabs in each replicate. They were artificially fed to satiation and fed once every evening at 6:00 PM for a total of 8 weeks.

[0031] After the breeding experiment, the growth performance indicators, oocyte size, and cholesterol deposition in the hepatopancreas and gonads of Chinese mitten crabs were measured.

[0032] Preferably, the Chinese mitten crab in the rearing stage is purchased from a local farm of the Pukou Aquatic Teaching and Research Base of Nanjing Agricultural University. During the breeding experiment, 1 / 3 of new water is injected every day to maintain the water temperature at 23-29°C, the dissolved oxygen is greater than 5 mg / L, and the pH is between 7.3-8.4.

[0033] Preferably, the commercially available Chinese mitten crab feed has a protein content of 360 g / kg and a lipid content of 80 g / kg; the basal diet in experimental group 2 is prepared with fish meal, soybean meal, cottonseed meal, rapeseed meal and peanut meal as protein sources, soybean oil and fish oil as fat sources, and α-starch as a sugar source. The basal diet is made into particles with a diameter of 2.5 mm using a meat grinder, ventilated and dried, and stored at -20°C.

[0034] Preferably, the basal diet comprises 25% fish meal, 8% soybean meal, 3% cottonseed meal, 2.5% rapeseed meal, 27% peanut meal, 21.6% α-starch, 3.4% soybean oil, 1.2% fish oil, 2% sodium carboxymethyl cellulose, 2.2% calcium dihydrogen phosphate, 0.4% squalene, 0.4% zeolite powder, 1% premix and 2.3% mixture, wherein the mixture contains the following substances: 4.75% choline chloride; 1.72% antioxidant; 2.35% antifungal; 22.06% salt; 59.30% Lukangyuan; and 9.82% synbiotics.

[0035] The beneficial effects of the present invention are:

[0036] (1) Compared with commercially available formulated feeds, the method provided by the present invention can significantly improve the weight gain rate and gonadal index of Chinese mitten crabs during the growing period, increase the oocyte volume of Chinese mitten crabs during the growing period, make the yolk protein of Chinese mitten crabs more abundant, and improve the ovary quality during the growing period. This is of great significance for realizing the application of formulated feeds throughout the breeding process of Chinese mitten crabs.

[0037] (2) Compared with the existing commercially available Chinese mitten crab feed and chilled fish farming, by adding 0.2% to 0.4% squalene to the basic formula of Chinese mitten crab feed, the poor growth and gonadal development of Chinese mitten crab during the rearing period can be effectively improved, basically achieving the effect of chilled fish farming.

[0038] (2) The present invention provides a research method for evaluating squalene as a cholesterol substitute. When evaluating the replacement effect of squalene on cholesterol, a linear model of feed cholesterol content and hepatopancreatic cholesterol deposition in Chinese mitten crab is established to evaluate the biological efficacy of squalene in Chinese mitten crab farming, thereby more intuitively presenting the replacement effect of squalene as a cholesterol substitute. This method can be used as an effective method for screening cholesterol substitutes. The above method is not only scientific but also has wide applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the cholesterol deposition in the hepatopancreas of juvenile Chinese mitten crabs fed with different diets.

[0040] Figure 2 This is a linear model diagram of the hepatopancreas cholesterol deposition amount of juvenile Chinese mitten crabs fed with different cholesterol levels.

[0041] Figure 3 This is a HE staining diagram of the ovaries of Chinese mitten crabs in the growing period of the present invention fed with different diets, in which A is the control group; B is the experimental group 1; and C is the experimental group 2.

[0042] Figure 4 This is a schematic diagram of the cholesterol deposition in the hepatopancreas and gonads of the Chinese mitten crab during the growing period of the present invention when fed with different diets. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] Example 1 A method for studying squalene as a cholesterol substitute

[0045] 1. Experimental Design

[0046] The experimental feeds were divided into 7 groups:

[0047] (1) Control group: fed with basic feed;

[0048] (2) Experimental group 1: fed with a basal diet containing 0.2% cholesterol;

[0049] (3) Experimental group 2: fed with a basal diet containing 0.4% cholesterol;

[0050] (4) Experimental group 3: fed with a basic feed containing 0.05% squalene;

[0051] (5) Experimental group 4: fed with a basic feed containing 0.1% squalene;

[0052] (6) Experimental group 5: fed with a basic feed containing 0.2% squalene;

[0053] (7) Experimental Group 6: The rats were fed with a basic feed containing 0.4% squalene.

[0054] The raw material formula of each group of feed is shown in Table 1.

[0055] The basal diet was formulated with fish meal, soybean meal, cottonseed meal, rapeseed meal, and peanut meal as protein sources, soybean oil and fish oil as fat sources, and α-starch as a carbohydrate source. Pellets with a diameter of 2.5 mm were minced using a meat grinder, dried under ventilation, and stored at -20°C.

[0056] Table 1 Raw material formula of each test group

[0057]

[0058]

[0059] Note: The compound premix provides the following minerals (g / kg feed) and vitamins (IU or mg / kg feed): CuSO4·5H2O, 2g; FeSO4·7H2O, 25g; ZnSO4·7H2O, 22g; MnSO4·4H2O, 7g; Na2SeO3, 0.04g; KI, 0.026g; CoCl2·6H2O, 0.1g; Vitamin A, 900,000 IU; Vitamin D, 200 ,000 IU; Vitamin E, 4500 mg; Vitamin K3, 220 mg; Vitamin B1, 320 mg; Vitamin B2, 1090 mg; Vitamin B5, 2000 mg; Vitamin B6, 500 mg; Vitamin B12, 1.6 mg; Vitamin C, 10,000 mg; Pantothenic acid, 1000 mg; Folic acid, 165 mg; Choline, 60,000 mg; Biotin, 100 mg; Inositol 15,000 mg. The mixture contains the following substances (%): Choline chloride 4.75%; Antioxidant 1.72%; Antifungal agent 2.35%; Salt 22.06%; Green Kangyuan 59.30%; Synbiotic 9.82%.

[0060] 2. Experimental Crabs and Breeding Management

[0061] Juvenile Chinese mitten crabs (ERCs) were purchased from a local farm at the Pukou Fisheries Teaching and Research Base of Nanjing Agricultural University. The experiment was conducted outdoors in white barrels (1.07 × 0.8 × 0.34 m, L:W:H). The crabs were acclimated for two weeks, fed a commercial diet (360 g / kg protein and 80 g / kg lipids). After acclimation, 420 male Chinese mitten crabs of good physique, uniform size, and an initial weight of 5.6 ± 0.05 g were selected. Each group consisted of four replicates, with 15 crabs per replicate. They were fed to satiation, with a single feeding at 6:00 PM daily for eight weeks. During the experiment, residual bait was removed daily, and the number of molts and deaths was counted. One-third of the water was refilled daily to maintain a water temperature of 23-29°C, dissolved oxygen greater than 5 mg / L, and a pH between 7.3 and 8.4.

[0062] 3. Test data measurement and result analysis

[0063] 3.1 Determination of growth performance

[0064] After the experiment, each group of Chinese mitten crabs was weighed, and the specific growth rate, hepatopancreas index, shelling rate, and molting rate were calculated using the following formulas. The results are shown in Table 2.

[0065] Hepatopancreatic index (HSI, %) = 100 × H f / W f ;

[0066] Specific growth rate (SGR, % / d) = 100 × (ln Wf - ln Wi) / t;

[0067] Meat yield = crab muscle weight / crab body weight

[0068] Molting rate = number of molts / number of survivors × 100%.

[0069] Wi and Wf are the initial and final average body weights of juvenile crabs, respectively; t is the experimental time; W0 and Wt are the initial and final total weights of juvenile crabs; Hf is the average weight of the hepatopancreas of juvenile crabs; L is the shell width.

[0070] As shown in Tables 2-1 and 2-2, cholesterol supplementation significantly affected the weight gain rate and specific growth rate of juvenile Chinese mitten crabs (p<0.05), but had no significant effect on survival rate or hepatopancreatic index (p>0.05). Juvenile Chinese mitten crabs achieved the highest weight gain rate when cholesterol supplemented at 0.2% in the diet. Supplementation of squalene significantly affected the weight gain rate, specific growth rate, and hepatopancreatic index of juvenile Chinese mitten crabs (p<0.05), but had no significant effect on survival rate or molting rate (p>0.05). Juvenile Chinese mitten crabs achieved the highest weight gain rate when squalene supplemented at 0.4% in the diet.

[0071] Table 2-1 Growth performance of groups with different cholesterol contents

[0072]

[0073] Table 2-2 Growth performance of groups with different squalene content

[0074]

[0075] like Figure 1 As shown in the results, the addition of cholesterol had a significant effect on the cholesterol deposition in the hepatopancreas of juvenile Chinese mitten crabs (p<0.05). The cholesterol deposition in the hepatopancreas increased with the increase of cholesterol content in the feed. Figure 2 The linear model of the cholesterol content in the feed and the cholesterol deposition in the hepatopancreas of Chinese mitten crabs is shown in Figure 2. The linear model shows that the amount of cholesterol added to the feed is positively correlated with the cholesterol deposition in the hepatopancreas of Chinese mitten crab juveniles.

[0076] like Figure 1 As shown in Table 3, the addition of 0.2% and 0.4% squalene to the feed significantly affected cholesterol deposition in the hepatopancreas of juvenile Chinese mitten crabs (p < 0.05). This revealed a correlation between squalene content in the feed and cholesterol deposition in the hepatopancreas of Chinese mitten crabs. Furthermore, the biological efficacy of squalene in replacing cholesterol at different supplemental levels ranged from 41% to 108%, as shown in Table 3.

[0077] The results show that if Figure 3 The results show that adding 0.05% to 0.4% squalene to the feed promoted cholesterol deposition in the hepatopancreas of juvenile Chinese mitten crabs. Adding 0.4% squalene had the greatest growth-promoting effect, while adding 0.2% to 0.4% squalene resulted in the highest hepatopancreatic cholesterol deposition. Squalene can effectively replace cholesterol in feed.

[0078] Table 3 Biological efficacy of squalene in different amounts as a substitute for cholesterol

[0079]

[0080] Table 3 shows that the squalene content in the feed and its corresponding bioavailability show a negative correlation. Specifically, as the squalene content in the feed increases, the bioavailability gradually decreases. Consequently, the utilization rate of squalene by juvenile Chinese mitten crabs decreases with increasing squalene intake. These results suggest that there are certain limits to the utilization rate of squalene in the feed by juvenile Chinese mitten crabs. In practical production applications, it is necessary to balance the amount of squalene used and its corresponding bioavailability to maximize benefits.

[0081] Example 2 A method for promoting the growth and gonadal development of Chinese mitten crabs during the breeding period

[0082] 1. Experimental Design

[0083] The experimental feeds were divided into three groups:

[0084] (1) Control group: fed with commercially available Chinese mitten crab feed;

[0085] (2) Experimental group 1: fed with fresh ice fish;

[0086] (3) Experimental Group 2: The rats were fed a basal diet containing 0.4% squalene. The basal diet formula for Experimental Group 2 is shown in Table 4.

[0087] Because traditional icefish feeding has many drawbacks, the team has been working to replace fresh-frozen fish with formulated feed. The purpose of feeding icefish in Experimental Group 2 was to use icefish as a reference to determine the effectiveness of the formulated feed. The goal was to determine whether the formulated feed plus squalene could achieve the same results as fresh-frozen fish.

[0088] The basal diet was formulated with fish meal, soybean meal, cottonseed meal, rapeseed meal, and peanut meal as protein sources, soybean oil and fish oil as fat sources, and α-starch as a carbohydrate source. Pellets with a diameter of 2.5 mm were minced using a meat grinder, dried under ventilation, and stored at -20°C.

[0089] Table 4 Basic diet raw material formula of experimental group 2

[0090]

[0091]

[0092] Note: The compound premix provides the following minerals (g / kg feed) and vitamins (IU or mg / kg feed): CuSO4·5H2O, 2g; FeSO4·7H2O, 25g; ZnSO4·7H2O, 22g; MnSO4·4H2O, 7g; Na2SeO3, 0.04g; KI, 0.026g; CoCl2·6H2O, 0.1g; Vitamin A, 900,000 IU; Vitamin D, 200 ,000IU; Vitamin E, 4500mg; Vitamin K3, 220mg; Vitamin B1, 320mg; Vitamin B2, 1090mg; Vitamin B5, 2000mg; Vitamin B6, 500mg; Vitamin B12, 1.6mg; Vitamin C, 10,000mg; Pantothenic acid, 1000mg; Folic acid, 165mg; Choline, 60,000mg; Biotin, 100mg; Inositol 15,000mg.

[0093] The mixture contains the following substances (%): choline chloride 4.75%; antioxidant 1.72%; mildew inhibitor 2.35%; salt 22.06%; Lukangyuan 59.30%; synbiotic 9.82%.

[0094] 2. Experimental crabs and breeding management

[0095] The experimental subjects were Chinese mitten crabs (Eriocheir sinensis) in the rearing stage, purchased from a local farm at the Pukou Fisheries Teaching and Research Base of Nanjing Agricultural University. The breeding experiment was conducted in an outdoor cement tank (1×1×0.8m, L:W:H). The experimental crabs were trained for 2 weeks, during which time they were fed a commercial feed (360g / kg protein and 80g / kg lipid). After acclimation, 120 female Chinese mitten crabs with a robust physique, uniform size, and an initial weight of (70.37±0.47g) were selected. Four replicates of 10 crabs were selected for each group. They were artificially fed to satiation and fed once every evening at 6:00 PM for a total of 8 weeks. During the experimental period, residual bait was removed daily, and the number of molts and deaths was counted. One-third of the water was added daily to maintain the water temperature at 23-29°C, the dissolved oxygen greater than 5mg / L, and the pH between 7.3 and 8.4.

[0096] 3. Test data measurement and result analysis

[0097] 3.1 Determination of growth performance

[0098] After the experiment, the three groups of Chinese mitten crabs were weighed, and the weight gain rate, survival rate, hepatopancreas index, gonad index, and fatness were calculated. The calculation formula is as follows, and the results are shown in Table 5.

[0099] Weight gain rate (WGR, %) = 100 × (W f -W i ) / W i ;

[0100] Hepatopancreatic index (HSI, %) = 100 × H f / W f ;

[0101] Gonadal index (GSI, %) = 100 × G f / W f ;

[0102] Fatness (K) = W t / L 3 ;

[0103] Wi and Wf are the initial and final average body weights of Chinese mitten crab, respectively; Hf is the average weight of the hepatopancreas of Chinese mitten crab; Gf is the average weight of the gonads of Chinese mitten crab; Wt is the total weight of Chinese mitten crab at the end; L is the shell width.

[0104] Table 5 Growth performance test table of each group of Chinese mitten crab

[0105]

[0106] As shown in Table 5, compared with the control group, experimental groups 1 and 2 had significant effects on the final weight, weight gain rate, hepatopancreas index, and gonadal index of the Chinese mitten crab during the rearing period (P < 0.05). The weight gain rate of experimental group 1 was significantly higher than that of experimental group 2 (P < 0.05), but there was no significant difference in the gonadal index (P > 0.05). These results indicate that the addition of 0.4% squalene to a formula feed can promote the growth and gonadal development of Chinese mitten crab during the rearing period, achieving similar effects as feeding fresh-frozen fish. Therefore, feeding a formula feed with 0.4% squalene can replace fresh-frozen fish.

[0107] 3.2 Ovarian histological analysis

[0108] After the experiment, the size of the oocytes was measured, and the effect of squalene on the size of Chinese mitten crab oocytes was determined based on these parameters.

[0109] Ovarian tissue was fixed in 4% paraformaldehyde for 24 h and then dehydrated in a gradient of 70%, 75%, 80%, 90%, 95%, and anhydrous ethanol. Afterwards, the tissue was cleared with xylene and embedded in paraffin. The embedded ovarian tissue was cut into 6 mm slices using a microtome (LEICA 2016, Berlin, Germany). The sections were then dewaxed and rehydrated in anhydrous ethanol, 95%, and 80% anhydrous ethanol, respectively. After rehydration, the sections were immersed in ultrapure water for 5 min and then stained with hematoxylin and eosin (H&E). Finally, the sections were sealed with neutral gum and observed using a virtual microscope (Nikon, Tokyo, Japan). Images were taken at 20x magnification (Nikon, DS-U2, Tokyo, Japan). Nuclear and oocyte parameters were measured using Image-Pro Plus 6.0 (Americamedia Cybernetics). These parameters include the long diameter of the oocyte (LO), the long diameter of the nucleus (LN), the area of the oocyte and the area of the corresponding yolk granules. Finally, the volume of the oocyte (VO), the volume of the nucleus (VN) and the area ratio of the yolk granules in the oocyte were calculated according to the formula disclosed in Wu X, Liu M, Pan J, et al. The ovarian development pattern of pond-reared Chinese mitten crab, Eriocheir sinensis H. Milne-Edwards, 1853 [J]. Crustaceana, 2017, 90 (4): 449-470. (Wu et al., 2017), and the area ratio of the yolk granules in the oocyte are as follows:

[0110] The volume of the oocyte (VO) = 0.523 × Wo2 × Lo;

[0111] The volume of the nucleus (VN) = 0.523 × Wn2 × Ln;

[0112] Yolk granule area ratio = yolk granule area / oocyte area.

[0113] Wo: maximum width of oocyte; Lo: maximum length of oocyte; Wn: maximum width of nucleus; Ln: maximum length of nucleus

[0114] The results are shown in Table 6:

[0115] Table 6 Data on the size of Chinese mitten crab oocytes in each group during the rearing period

[0116]

[0117]

[0118] Table 3 shows the parameters of Chinese mitten crab oocytes during the rearing period. Compared with the control group, experimental group 2 showed significant differences in oocyte length, oocyte volume, and yolk granule area ratio (p < 0.05). Although experimental group 2 showed significant differences in oocyte length, oocyte volume, and nuclear volume compared with experimental group 1 (p < 0.05), there was no significant difference in yolk granule area ratio between experimental group 2 and experimental group 1 (p > 0.05).

[0119] like Figure 3 As shown in the figure, compared with the control group, the oocytes in experimental group 2 were larger and more squeezed and deformed, and the oocytes were filled with yolk granules. Further observation revealed that although the oocytes in experimental group 2 were smaller than those in experimental group 1, the yolk granules in the oocytes in experimental group 2 were more filled.

[0120] These results indicate that supplementing feed with 0.4% squalene significantly improved growth performance and gonadal indices, as well as oocyte volume and the area ratio of yolk granules in oocytes, during the grower period compared to a commercially available feed. Compared to the chilled fish group, the commercially available feed significantly increased the hepatopancreas index, and 0.4% squalene promoted gonadal development in Chinese mitten crabs. This confirms the importance of squalene for ovarian development in Chinese mitten crabs during the grower period.

[0121] 3.3 Determination of cholesterol content

[0122] The cholesterol content in tissues was determined using high-performance liquid chromatography (HPLC). First, 1 g of sample was weighed and placed in a 50 mL centrifuge tube. 15 mL of anhydrous ethanol and 5 mL of 60% potassium hydroxide were then added and saponified in a 90°C water bath for 1 hour. The sample was then centrifuged at 5000 g for 5 minutes. 1 mL of the supernatant was taken and made up to 10 mL with anhydrous ethanol before determination. The HPLC instrument used in the experiment was a Thermo U3000, and the chromatographic column used was an Agilent ZORBAX Eclipse Plus column C18 5 μm 4.6 × 150 mm. 100% methanol was used as the mobile phase at a flow rate of 1 mL / min.

[0123] like Figure 4 The results showed that adding 0.4% squalene to feed significantly increased the cholesterol content in the hepatopancreas of Chinese mitten crabs during the grow-out period, compared to a commercially available feed. While there was no significant difference in gonadal cholesterol content per unit weight, the total amount of gonadal cholesterol in the fresh-frozen fish group and the group supplemented with 0.4% squalene was higher than that in the commercially available feed. This also confirms the importance of squalene in regulating cholesterol deposition in the Chinese mitten crabs during the grow-out period.

[0124] In summary, the present invention evaluated the biological efficacy of squalene in Chinese mitten crab farming by establishing a linear model between feed cholesterol content and hepatopancreatic cholesterol deposition in the Chinese mitten crab (Eriocheir sinensis). This method can be used as an effective method for screening cholesterol substitutes. Furthermore, compared with existing commercially available Chinese mitten crab feed and chilled fish farming, adding 0.4% squalene to the basic formula of Chinese mitten crab feed can effectively improve the poor growth and gonadal development of Chinese mitten crabs during the rearing period, essentially achieving the same results as chilled fish farming.

[0125] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0126] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A research method for replacing cholesterol in Chinese mitten crab feed, characterized by: The experimental design is as follows: The experiment was divided into seven groups: (1) Control group: fed with basic feed; (2) Experimental group 1: fed with a basal feed containing 0.2% cholesterol; (3) Experimental group 2: fed with a basal diet containing 0.4% cholesterol; (4) Experimental group 3: fed with a basic feed containing 0.05% squalene; (5) Experimental group 4: fed with a basic feed containing 0.1% squalene; (6) Experimental group 5: fed with a basic feed containing 0.2% squalene; (7) Experimental group 6: fed with a basic feed containing 0.4% squalene; The experimental crabs were trained for 2 weeks and fed with commercially available Chinese mitten crab feed. After acclimation, 420 strong, well-proportioned male Chinese mitten crabs with an initial weight of 5.6±0.05g were selected. Each group had four replicates, with 15 crabs in each replicate. They were fed artificially to satiation and fed once every evening at 18:00 for a total of 8 weeks. The commercially available Chinese mitten crab feed has a protein content of 360 g / kg and a lipid content of 80 g / kg. The basal diet is prepared with fish meal, soybean meal, cottonseed meal, rapeseed meal, and peanut meal as protein sources, soybean oil and fish oil as fat sources, and α-starch as a sugar source. The basal diet is made into pellets with a diameter of 2.5 mm using a meat grinder, dried under ventilation, and stored at -20°C. After the breeding experiment, the growth performance of Chinese mitten crabs in different cholesterol and squalene content groups and the hepatopancreatic cholesterol deposition of juvenile Chinese mitten crabs fed with different diets were measured. A linear model of the hepatopancreatic cholesterol deposition of juvenile Chinese mitten crabs fed with different cholesterol levels was constructed, and the biological efficacy of different amounts of squalene added to replace cholesterol was analyzed. The results showed that the biological efficacy of squalene in replacing cholesterol at different addition amounts ranged from 41% to 108%; Adding 0.05% to 0.4% squalene to feed can promote the deposition of cholesterol in the hepatopancreas of juvenile Chinese mitten crabs. Adding 0.4% squalene has the best growth-promoting effect, and adding 0.2 to 0.4% squalene has the highest hepatopancreatic cholesterol deposition.

2. The research method for replacing cholesterol in Chinese mitten crab feed according to claim 1, characterized in that: The experimental crabs were juvenile Chinese mitten crabs, purchased from a local farm at the Pukou Fisheries Teaching and Research Base of Nanjing Agricultural University. During the breeding experiment, 1 / 3 of new water was injected every day to maintain the water temperature at 23-29°C, the dissolved oxygen greater than 5 mg / L, and the pH between 7.3-8.

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3. The research method for replacing cholesterol in Chinese mitten crab feed according to claim 1, characterized in that: The steps for preparing the linear model of hepatopancreatic cholesterol deposition of juvenile Chinese mitten crabs fed with different cholesterol levels are as follows: S1. A linear model was developed to determine the relationship between dietary cholesterol content and hepatopancreatic cholesterol deposition in Chinese mitten crabs by feeding them diets containing 0%, 0.2%, and 0.4% cholesterol. S2. The correlation between squalene content in the feed and cholesterol deposition in the hepatopancreas of Chinese mitten crabs was determined by feeding them feeds containing 0.05%, 0.1%, 0.2%, and 0.4% squalene. S3. Using the linear model established in S1, the biological efficacy of different amounts of squalene in replacing cholesterol was calculated to be 41% to 108%.

4. The research method for replacing cholesterol in Chinese mitten crab feed according to claim 1, characterized in that: The optimal addition range of squalene in feed is 0.2% to 0.4%.

5. A method for promoting gonadal development of Chinese mitten crab, characterized in that 0.4% squalene was added to the basal diet of Chinese mitten crab. The experimental design was as follows: The trial was divided into three groups: (1) Control group: fed with commercially available Chinese mitten crab feed; (2) Experimental group 1: fed with fresh ice fish; (3) Experimental group 2: fed with a basal diet supplemented with 0.4% squalene; The experiment selected Chinese mitten crabs in the rearing stage. The crabs were trained for 2 weeks and fed a commercially available Chinese mitten crab feed. After acclimation, 120 strong, well-proportioned female Chinese mitten crabs with an initial weight of 70.37±0.47g were selected. Each group had four replicates, with 10 crabs in each replicate. They were artificially fed to satiation and fed once every evening at 6:00 PM for a total of 8 weeks. The commercially available Chinese mitten crab feed had a protein content of 360 g / kg and a lipid content of 80 g / kg. The basal diet in experimental group 2 was prepared with fish meal, soybean meal, cottonseed meal, rapeseed meal, and peanut meal as protein sources, soybean oil and fish oil as fat sources, and α-starch as a sugar source. The basal diet was minced using a meat grinder to form pellets with a diameter of 2.5 mm, dried under ventilation, and stored at -20°C. After the breeding experiment, the growth performance indicators, oocyte size, and cholesterol deposition in the hepatopancreas and gonads of Chinese mitten crabs were measured.

6. The method for promoting gonadal development of Chinese mitten crab according to claim 5, wherein: During the culture test, 1 / 3 of the water was injected every day to maintain the water temperature at 23-29° C., the dissolved oxygen was greater than 5 mg / L, and the pH was between 7.3-8.

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7. The method for promoting gonadal development of Chinese mitten crab according to claim 5, characterized in that: The basic diet includes 25% fish meal, 8% soybean meal, 3% cottonseed meal, 2.5% rapeseed meal, 27% peanut meal, 21.6% α-starch, 3.4% soybean oil, 1.2% fish oil, 2% sodium carboxymethyl cellulose, 2.2% calcium dihydrogen phosphate, 0.4% squalene, 0.4% zeolite powder, 1% premix and 2.3% mixture, wherein the mixture contains the following substances: 4.75% choline chloride; 1.72% antioxidant; 2.35% anti-fungal agent; 22.06% salt; 59.30% Lukangyuan; and 9.82% synbiotics.

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