Application of defatted insect protein source in promoting ovary development of female parent shrimps

Abstract

The invention discloses application of defatted insect protein sources in promoting ovarian development of female parent shrimps, ovarian development of the female parent shrimps can be promoted to different degrees after three defatted insect protein sources are added into feed, especially after defatted silkworm chrysalis meal is added, the defatted insect protein sources can be used as main protein sources, and the ovarian development of the female parent shrimps can be promoted to different degrees. The serum sex hormone level of female parent shrimps is remarkably improved, lysine degradation, amino acid biosynthesis, and enrichment of metabolites of metabolic pathways related to amino acids and fatty acids, such as glycerophospholipids, arachidonic acid and 2-oxo-carboxylic acid metabolic pathways, are increased, and the protein content of hepatopancreas and the accumulation of triglyceride of ovaries are effectively improved; the development of ovaries is promoted and the development quality of ovaries is improved.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic feed, and in particular to application of a defatted insect protein source in promoting ovarian development of female broodstock. Background Art

[0002] Red claw crayfish (Cherax quadriarinatus), commonly known as Australian freshwater lobster, has the advantages of fast growth, large size and strong adaptability. It is considered to be one of the most promising commercial aquaculture species.

[0003] At present, there is no special compound feed developed for the nutritional enhancement stage of red claw crayfish parents on the market. In actual breeding production, shrimp feed is often combined with chilled animal protein sources such as earthworms, snails and minced fish, as well as plant protein sources such as cabbage, pumpkin and tender grass. However, this feeding method will lead to insufficient nutrition intake of female parents, and the ovarian tissue will not develop to the best state, and thus it will not be possible to provide a large number of stable seedlings.

[0004] It can be seen that the development of compound feed that meets the nutritional needs of female red claw crayfish is of great significance for promoting the gonadal development of red claw crayfish and improving reproductive performance. Summary of the invention

[0005] The invention provides a defatted insect protein source for use in promoting ovarian development of female broodstock, aiming to solve the problems existing in the above-mentioned background technology.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0007] On the one hand, a use of a defatted insect protein source in preparing a feed for promoting ovarian development of female broodstock is provided.

[0008] Preferably, the female broodstock comprises red claw crayfish (Cherax quadriarinatus).

[0009] Preferably, the defatted insect protein source comprises one or more of defatted Tenebrio molitor powder, defatted black soldier fly powder and defatted silkworm pupa powder, and by weight, the defatted mealworm powder has a crude protein content of 60-70% and a crude fat content of 8-10%, the defatted black soldier fly powder has a crude protein content of 45-55% and a crude fat content of 10-15%, and the defatted silkworm pupa powder has a crude protein content of 50-60% and a crude fat content of 10-18%.

[0010] On the other hand, a feed for promoting ovarian development of female broodstock is provided. The feed comprises, by weight, 100-200 g of fish meal, 150-200 g of defatted insect protein source, 145-180 g of microcrystalline cellulose, 150-200 g of gluten, 50-80 g of soybean oil, 0.3-1.0 g of 2,6-di-tert-butyl-4-methylphenol, 5-15 g of krill oil, 150-200 g of wheat starch, 4-6 g of cholesterol, 15-25 g of vitamin premix, 15-25 g of mineral premix, 1-5 g of choline chloride, 15-25 g of sodium carboxymethyl cellulose, 5-15 g of monocalcium phosphate, and 0.3-1.0 g of cuttlefish paste per kilogram, and the feed does not include soybean meal, cottonseed meal, and peanut meal.

[0011] Furthermore, every 100 g of the vitamin premix includes the following raw materials in parts by weight:

[0012] Vitamin B1 0.05 g; Vitamin B2 0.3 g; Vitamin B6 0.1 g; Vitamin B12 0.03 g; Vitamin C 10 g; Vitamin E 5 g; Vitamin K 0.2 g; Inositol 0.5 g; Niacinamide 0.5 g; Vitamin D3 (500000 IU / g) 0.08 g; Vitamin A (500000 IU / g) 0.05 g; Folic acid 0.02 g; Vitamin H 0.005 g; Choline chloride 10 g; Vitamin B 50.5 g; The rest are α-vitamins.

[0013] Furthermore, every 100 g of the mineral premix includes the following raw materials in parts by weight:

[0014] 2.8g potassium chloride; 7.1g magnesium sulfate; 2g zinc sulfate; 0.162g manganese sulfate; 0.06g copper sulfate pentahydrate; 0.02g calcium iodate; 0.01g cobalt chloride hexahydrate; 21.5g sodium phosphate; 25g calcium phosphate dihydrate; 18g calcium carbonate; 0.025g sodium selenite, 1.0g ferrous sulfate; 21.6g potassium hydrogen phosphate; the rest is α-vitamin.

[0015] On the other hand, there is also provided a use of the above feed in increasing the nutrient content of the hepatopancreas and / or ovary of female broodstock.

[0016] Preferably, the hepatopancreas nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose, and the ovarian nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose.

[0017] On the other hand, the invention also provides a use of the above feed in increasing the content of sex hormones in the serum of female broodstock.

[0018] Preferably, the sex hormone includes one or both of estradiol and progesterone.

[0019] On the other hand, the invention also provides an application of differential metabolites produced after feeding the above feed to female broodstock in detecting the ovarian development of female broodstock.

[0020] Preferably, the differential metabolites include one or more of linoleic acid, D-glutamine, D-glutamate, alanine, aspartic acid, glutamic acid, tryptophan, arginine and proline.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention can promote the ovarian development of female broodstock to different degrees by adding three defatted insect protein sources to the feed, especially by adding defatted silkworm pupa powder, which can be used as the main protein source, significantly improve the serum sex hormone level of female broodstock, increase the metabolite enrichment of metabolic pathways related to amino acids and fatty acids such as lysine degradation, amino acid biosynthesis, glycerophospholipid, arachidonic acid and 2-oxocarboxylic acid metabolic pathways, effectively increase the protein content of the hepatopancreas and the accumulation of triglycerides in the ovaries, promote the development of the ovaries, and improve the quality of ovarian development. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0024] Figure 1 Effects of defatted insect protein sources on liver-to-body ratio (A) and gonad index (B) of female red claw crayfish;

[0025] Figure 2 This is the lipid deposition in the hepatopancreas of female red claw crayfish after ORO staining. ORO dye dyes lipids in tissues red. The vertical axis represents the grouping, and the horizontal axis represents the magnification.

[0026] Figure 3 The developmental stages of the female red claw crayfish ovary after H&E staining, FC: follicular cell; C: ooplasm; Oo: oocyte; Y: yolk granule; N: nucleus; PVS: perivitelline space;

[0027] Figure 4 Effects of defatted insect protein source on the nutrient content of hepatopancreas of female redclaw crayfish: (A) total protein; (B) triglyceride; (C) total cholesterol; (D) glucose; (E) glycogen;

[0028] Figure 5 The effects of defatted insect protein source on the nutrient content of female red claw crayfish ovary, (A) total protein; (B) triglyceride; (C) total cholesterol; (D) glucose; (E) glycogen;

[0029] Figure 6 Effects of defatted insect protein sources on the serum sex hormone levels of female red claw crayfish: (A) estradiol; (B) progesterone; (C) methyl farnesate;

[0030] Figure 7 Results of differential metabolite screening of red claw crayfish serum in the BM group, (A) Metabolite classification and proportion diagram; (B) PCA model score scatter plot: the closer the scatter points (samples), the more similar the sample metabolites; (C) OPLS-DA model score scatter plot: the farther the horizontal distance of the scatter points (samples), the greater the difference between the groups, and the closer the vertical distance, the higher the repeatability within the group; (D) OPLS-DA model permutation test diagram: the horizontal axis represents the accuracy of the permutation test random model, and the vertical axis represents the number of random models; (E) Volcano diagram of differential metabolite screening: the chi-square standard is P<0.05, VIP>1; the larger the scatter point (peak), the larger the VIP value; metabolite level, red: significantly upregulated; blue: significantly downregulated; gray: no significant difference;

[0031] Figure 8 This is the KEGG classification diagram of differential metabolites in the BM group;

[0032] Fig. 9 This is the KEGG enrichment map of differential metabolites in the BM group. The horizontal axis is Rich Factor (the ratio of the number of differential metabolites in a certain pathway to the number of metabolites in the pathway), and the vertical axis is the metabolic pathway. The size of the dots represents the number of differential metabolites in the pathway.

[0033] The redder the dot, the smaller the P value;

[0034] Fig.10 This is the metabolic pathway analysis diagram of the BM group. The horizontal axis and the size of the bubble (metabolic pathway) represent the size of the influencing factor; the vertical axis and the color of the bubble represent the P value [-ln(p)] of the enrichment analysis. The darker the color, the smaller the P value. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] Example:

[0037] Experimental design and preparation of experimental feed

[0038] In the breeding experiment of the present embodiment, the selected female broodstock is red claw crayfish, purchased from the Guangxi Agricultural Variety Hainan South Breeding Base in Ledong Li Autonomous County, Hainan Province, China. This experiment is provided with 5 treatment groups, 4 parallels in each treatment group, 10 female broodstock in each parallel, wherein the fish meal (FM) group is the control group, the plant meal (VM) group is the traditional protein experimental group, and the experimental groups are respectively defatted yellow mealworm (TM) group, defatted black soldier fly powder (HM) group, defatted silkworm pupa powder (BM) group. Red claw crayfish needs to be temporarily raised for 5 days to adapt to the experimental environment conditions. During the temporary raising period, commercial pellet feed (such as Tongwei feed, etc.) is fed twice a day, and six-month-old female broodstock (40.00 ± 2.00g) with consistent specifications, health and complete appendages are randomly selected, and then randomly assigned to the basket with a tie to fix the foam rod, and 1 broodstock is put into each basket to avoid the crayfish killing each other during the experiment, thereby affecting the accuracy of the experiment. During the breeding experiment, 10 frames were set up in each parallel, and one female broodstock from each group was placed in each frame for breeding. The average temperature of the breeding water was 28.0±2.0℃, the average pH was 7.80±0.20, the dissolved oxygen was ≥6.0mg / L, and the ammonia nitrogen was <0.05mg / L.

[0039] In this embodiment, a total of 5 groups of equal nitrogen and equal fat feeds are provided. The feed ingredients and formulas are shown in Table 1, which correspond to feeding FM group, VM group, TM group, HM group and BM group of broodstock, respectively. The three feeds of TM, HM and BM in Table 1 are all feeds for promoting ovarian development of female broodstock in the present invention.

[0040] When preparing the experimental feed, first grind the fish meal, soybean meal, cottonseed meal and peanut meal and pass them through a 60-mesh sieve, and then weigh the raw materials according to the proportion shown in the feed composition table (Table 1). The powdered raw materials are mixed step by step from small to large in proportion, and then the oil source is added. The mixed raw materials are passed through a 60-mesh sieve, and then 120mL / kg of distilled water is added to the premix. Finally, the premix is ​​extruded using a cold-pressed double-screw plodder to make a feed with a particle size of 2.5mm. The feed particles are then dried in a well-ventilated environment until the moisture content of the feed is less than 10%, and then the feed is stored in a refrigerator at -20°C. After testing, the approximate composition of the 5 groups of experimental feeds is shown in Table 1.

[0041] Table 1 Five experimental feed formulations (g / kg dry weight) and approximate composition (% dry weight)

[0042]

[0043]

[0044] Among them, defatted mealworm powder, defatted black soldier fly powder and defatted silkworm pupa powder were all purchased from Haikun Agricultural Technology Co., Ltd.

[0045] Farming management and experimental conditions

[0046] The broodstock in the five treatment groups were cultured for 50 days and fed with the corresponding experimental feeds at fixed times every day. In each treatment group, the daily feed amount was 4% of the body weight of the red claw crayfish, and the feeding was carried out at 8:00 and 16:00 every day. The residual feed and feces were removed 2 hours after feeding.

[0047] In addition, in each treatment group, two-thirds of the water was replaced every three days to maintain the best water quality. During the culture period, the average temperature of the culture water in each group was 29.0±3.0℃, the average pH was 7.50±0.20, the dissolved oxygen was 6.0-7.5mg / L, and the ammonia nitrogen was <0.05mg / L.

[0048] Sample collection

[0049] Before the end of the breeding experiment, the female broodstock in each group were fasted for 24 hours, and the red claw crayfish were anesthetized in an ice bath. The surface moisture of the red claw crayfish was wiped dry and the final weight was measured. Hemolymph was extracted from the pericardial cavity of the red claw crayfish with a disposable 1mL syringe, and the hemolymph was placed in a 1.5mL centrifuge tube. After being placed at 4℃ for 24 hours, it was centrifuged at 4℃ and 3500rpm for 10 minutes to separate the serum sample for the detection of sex hormone content.

[0050] The cephalothorax of red claw crayfish was cut open with ophthalmic scissors, and the ovarian and hepatopancreatic tissues were quickly separated with tweezers. The hepatopancreas and ovaries of 4 red claw crayfish were randomly selected from each group and placed in a 4% paraformaldehyde solution for making tissue sections. The rest were placed in cryovials, quickly frozen in liquid nitrogen, and stored in a -80℃ refrigerator.

[0051] Phenotypic parameter statistics

[0052] The hepatosomatic index (HSI) and gonadal index (GSI) of the broodstock in each group were calculated according to the following formula:

[0053] Liver to body ratio (%) = 100% × hepatopancreas weight (g) / body weight (g)

[0054] Gonadal index (%) = 100% × ovarian weight (g) / body weight (g)

[0055] like Figure 1 As shown in the figure, the liver-to-body ratio of red claw crayfish in the TM, HM and BM groups was higher than that in the FM group, and the HM group had the highest ratio (P<0.05), but there was no significant difference between them and the VM, TM and BM groups ( Figure 1Part A, P>0.05), the gonadal index of red claw crayfish in TM and BM groups was significantly higher than that in VM, HM and control groups ( Figure 1 Part B, P < 0.05).

[0056] In the evaluation of crustacean broodstock, the liver-to-body ratio is usually used to evaluate the nutritional reserve of crustaceans, and the gonad index is used to evaluate the development of gonadal tissue. In this example, the three defatted insect protein sources had no significant negative effects on the liver-to-body ratio and gonad index of female red claw crayfish broodstock, among which the liver-to-body ratio of the red claw crayfish in the HM group was the highest, and the gonad index of the TM group and the BM group was significantly higher than that of the VM group, the HM group and the control group, indicating that the addition of defatted yellow mealworm powder, defatted black soldier fly powder, and defatted silkworm pupa powder to the feed can improve the nutritional reserve of broodstock.

[0057] Histological observation

[0058] The left upper half of the hepatopancreas of each group of broodstock obtained by sampling is uniformly selected for making tissue sections, and the sections are stained by Oil red O (ORO) staining method, which includes the following steps:

[0059] The hepatopancreatic tissue samples were fixed in a 4% paraformaldehyde solution, then placed in a 200 g / L sucrose-PB solution for 24 h, taken out and placed in an embedding medium, and stored at -80°C.

[0060] The tissue block was sliced ​​with a microtome (thickness of about 8 μm) and stored at -20°C until use;

[0061] When staining is required, the slices were placed at room temperature for 20 minutes, dried naturally, and then stained using the ORO staining method. After washing away the floating color, the staining effect was examined under a microscope. The lipid droplets were orange-red to bright red, and the cell nuclei were blue. The slices were sealed with glycerol gelatin and finally observed and photographed under a microscope (Eclipse 200, Nikon, Japan).

[0062] The ovarian tissues of each group of broodstock obtained by sampling uniformly selected the upper left half of the ovary for making tissue sections, and the hematoxylin-eosin (H&E) staining method was used, which includes the following steps:

[0063] The ovarian tissue samples were fixed in 4% paraformaldehyde solution, washed with distilled water, and then transferred to 0%, 80%, 90%, and 100% ethanol solutions for dehydration. The ovarian tissue samples were then transferred to xylene and subsequently embedded in paraffin;

[0064] The ovarian tissue samples were sliced ​​with a slicer (thickness of about 5 μm) and stored at -20°C for later use;

[0065] When staining is required, the sections were placed at room temperature for 20 minutes, dried naturally, and then stained using the H&E staining method. After washing away the floating color, the sections were sealed with neutral gum. The staining effect was observed under a microscope, and the cell nucleus was blue and the cytoplasm was red. Finally, the sections were observed and photographed under a microscope (Eclipse 200, Nikon, Japan).

[0066] like Figure 2 As shown, the lipid deposition in the hepatopancreas of red claw crayfish in the BM and HM groups was similar to that in the control group. Oil Red O dye made the red spots of hepatopancreas lipid in female shrimps in the BM and HM groups darker and denser, while the hepatopancreas lipid accumulation in the red claw crayfish in the TM group was relatively less.

[0067] like Figure 3 As shown in the figure, the ovaries of red claw crayfish in the BM group were in the late stage of development, the oocytes were enlarged and squeezed each other, showing an elliptical or polygonal cell morphology, in which the yolk granules and lipid droplets were denser, and the nuclei disappeared. The ovaries of red claw crayfish in the HM group were mostly in the middle stage of development, with clearly visible nuclei. The ovaries of red claw crayfish in the TM group, VM group and control group were in the early or middle stage of development, the oocytes developed asynchronously, and the perivitelline space was larger.

[0068] Determination of nutrients and sex hormone content

[0069] Two hepatopancreatic tissue samples and two ovarian tissue samples were randomly selected from each parallel group.

[0070] Take 0.1g of each hepatopancreatic tissue sample and prepare a 10% (mass volume ratio (w / v)) tissue homogenate with 0.9mL of pre-cooled 0.85% saline. Centrifuge for 15min at 4°C and 3500rpm, and retain the supernatant for detecting the nutrient content in the hepatopancreatic tissue, wherein the nutrient content in the hepatopancreatic tissue includes one or more of total protein, triglycerides, total cholesterol, glycogen, and glucose.

[0071] The ovarian tissue samples were processed in the same way as the hepatopancreatic tissue samples described above, and the total protein, triglyceride, total cholesterol, glycogen, and glucose contents were also tested.

[0072] The above indicators of hepatopancreas and ovary were detected by commercial detection kits purchased from Nanjing Jiancheng Bioengineering Institute. The detection steps were performed according to the instructions of the kits, and finally the absorbance was measured by an ELISA reader (Epoch, BioTek, USA).

[0073] Two serum samples were randomly taken from each parallel group and tested for sex hormone content, wherein the sex hormone content includes one or more of estradiol, progesterone and methyl farnesoate.

[0074] Estradiol, progesterone and methylfarnesoate in serum were detected using Shanghai Fantai Biological ELISA kit, and the detection steps were carried out according to the instructions.

[0075] like Figure 4 As shown in Figure 2, the total protein content of the hepatopancreas of red claw crayfish in the BM group was the highest ( Figure 4 The triglyceride content of the hepatopancreas of red claw crayfish in the HM group was significantly higher than that in the VM and BM groups ( Figure 4 The total cholesterol content in the B part of the TM group was not significantly different from that in the control group (P<0.05), but was significantly higher than that in the VM and BM groups ( Figure 4 The glucose content in the hepatopancreas of red claw crayfish in VM group was significantly higher than that in TM group ( Figure 4 The glycogen content of the hepatopancreas of the red claw crayfish in the control group was the lowest ( Figure 4 (Part E, P < 0.05).

[0076] like Figure 5 As shown in Figure 2, the total protein content of the red claw crayfish ovaries in the TM, HM, and BM groups was higher than that in the VM and FM groups (P<0.05), but there was no significant difference between the TM group and the other treatment groups ( Figure 5 The triglyceride content in the ovaries of red claw crayfish in the TM, HM and BM groups was significantly higher than that in the VM and control groups ( Figure 5 The total cholesterol content in the ovaries of red claw crayfish in the HM group was significantly higher than that in the control group (P<0.05), while the total cholesterol content in the TM and BM groups was not significantly different from that in the VM group ( Figure 5 The glucose content in the ovaries of red claw crayfish in VM group was significantly higher than that in control group (P<0.05), but there was no significant difference with other treatment groups ( Figure 5 There was no significant difference in the glycogen content of the ovaries of red claw crayfish in each group ( Figure 5 (Part E, P>0.05).

[0077] Insufficient nutrient reserves in the hepatopancreas of crustaceans will affect the quantity and quality of nutrient resources allocated to embryos by females, thereby affecting the quality of offspring. During folliculogenesis, lipids, proteins and carbohydrates are transferred from the hepatopancreas to the ovaries through blood lipids, and the proteins accumulated in the ovaries provide amino acids for larval development. In this embodiment, the total protein content of the hepatopancreas of red claw crayfish in the BM group reached twice that of the total protein content of other treatment groups, and the total protein content in the ovaries was not lower than that of other groups. At the same time, there was no significant difference in total protein content between the TM group and the HM group and the control group, indicating that the addition of defatted mealworm powder and defatted black soldier fly powder will not have a negative impact on the ovarian development of parent shrimp.

[0078] Therefore, defatted mealworm powder, defatted black soldier fly powder and defatted silkworm pupa powder can be added to the feed, and the addition of defatted silkworm pupa powder can significantly improve the protein nutrition level of the hepatopancreas and ovaries, and provide sufficient protein for the transport and deposition of protein substances in the ovaries.

[0079] Lipids are carriers of fat-soluble vitamins, can be used as energy sources, and provide fatty acids and phospholipids to maintain the growth, reproduction and health of aquatic animals. Lipids in the hepatopancreas are transported to the ovaries through blood lipids, which plays an important role in the reproductive process. The lipids in the hepatopancreas are mainly composed of triglycerides, cholesterol and phospholipids, and the high ratio of triglycerides to cholesterol is a good indicator for evaluating the nutritional status of animals.

[0080] In this example, although the triglyceride and total cholesterol content of the hepatopancreas of the red claw crayfish in the BM group was low, in the hepatopancreas tissue sections stained with Oil Red O, the lipid deposition amount of the hepatopancreas of the red claw crayfish in the BM group was similar to that of the red claw crayfish in the HM group, which had the highest triglyceride and cholesterol content. In addition, the triglyceride and total cholesterol content of the ovary of the red claw crayfish in the BM group was not significantly different from that of the TM group and the HM group. This shows that although there is a statistical difference between the lipid content of the hepatopancreas in the BM group and the HM group, the lipid accumulation of the ovary in the BM group is not affected by the lipid level of the hepatopancreas.

[0081] On the other hand, triglycerides in the hepatopancreas are the main energy source for crustacean physiological metabolism. Ovarian development requires a large amount of triglycerides for biosynthesis. In the ovarian tissue sections, the maturity of the ovarian development of red claw crayfish in the BM group was higher. It is speculated that the rapid development of the ovaries in the BM group consumed more triglycerides. In addition, the levels of estradiol and progesterone in the serum of red claw crayfish in the BM group were higher than those in other treatment groups. Cholesterol is a precursor of sex steroid hormones (such as estradiol and progesterone). Cholesterol from the hepatopancreas will also be used more for the synthesis of sex steroid hormones, promoting the development of the ovaries.

[0082] like Figure 6As shown in Figure 2, the estradiol content in the serum of red claw crayfish in the BM group was significantly higher than that in each treatment group (P<0.05), and the estradiol content in the serum of red claw crayfish in the HM and TM groups was also significantly higher than that in the control group ( Figure 6 The progesterone content in the serum of red claw crayfish in the BM group was significantly higher than that in the VM group, TM group and control group ( Figure 6 There was no significant difference in serum methyl farnesate content among the groups of red claw crayfish ( Figure 6 (Part C, P>0.05).

[0083] The levels of estradiol and progesterone in crustacean parents play a regulatory role in the development of gonads, and their physiological functions include regulating follicle formation, oocyte development and ovarian maturation. In this example, the estradiol and progesterone levels in the serum of red claw crayfish in the VM group, TM group and BM group were higher than those in the control group (and the levels in the BM group were the highest), indicating that adding defatted mealworm powder, defatted black soldier fly powder and defatted silkworm pupa powder to the feed can increase the levels of estradiol and progesterone in the serum.

[0084] On the other hand, in the ovarian tissue sections, the ovaries of red claw crayfish in the BM group were in the late stage of development, the ovaries of red claw crayfish in the HM group were in the middle stage of development, and the ovaries of red claw crayfish in the TM group, VM group and control group were in the early or middle stage of development. Combining the results of serum sex hormone content and ovarian tissue sections, it can be concluded that adding defatted silkworm pupa powder to feed can especially increase the serum estradiol and progesterone levels of crayfish and promote the development and maturation of the ovaries.

[0085] Amino acid composition analysis of feed, hepatopancreas and ovarian tissues

[0086] The corresponding experimental feed of each group was placed in 6 mol / L hydrochloric acid, hydrolyzed at 110°C, filtered and evaporated, then mixed with 0.05 mol / L hydrochloric acid, and impurities were filtered out with a 0.2 μm nylon membrane.

[0087] Phenyl isothiocyanate was used as the raw material to generate phenylthiocarbamate through a pre-column derivatization reaction, and the amino acid composition was analyzed on an automatic amino acid analyzer (Biochrom 20, Germany). The results are shown in Table 2.

[0088] The sample processing and amino acid composition analysis methods of hepatopancreas and ovarian tissues were the same as those of the above-mentioned experimental feeds. The amino acid compositions of hepatopancreas and ovary are shown in Tables 3 and 4, respectively.

[0089] Table 2 Amino acid composition of five experimental feeds (%)

[0090]

[0091]

[0092] Note: Asp (Aspartic acid), Thr (Threonine), Ser (Serine), Glu (Glutamic acid), Gly (Glycine), Ala (Alanine), Val (Valine), Met (Methionine), Ile (Isoleucine), Leu (Leucine), Tyr (Tyrosine), Phe (Phenylalanine), His (Histidine), Lys (Lysine), Arg (Arginine).

[0093] Table 3 Amino acid composition of hepatopancreas of five groups of female red claw crayfish

[0094]

[0095] Table 4 Amino acid composition of ovaries of five groups of female red claw crayfish

[0096]

[0097]

[0098] As shown in Table 3, the amino acid composition of the hepatopancreas of red claw crayfish in the BM group was significantly lower than that of the control group except for threonine, methionine and lysine (P<0.05), while the other amino acid composition ratios were not significantly different from those of the control group (P>0.05). The ratios of valine, isoleucine, tyrosine and phenylalanine in the hepatopancreas of the TM group were not significantly different from those of the control group (P>0.05), but the ratios of the other amino acids were significantly lower than those of the control group (P<0.05). The ratios of each amino acid in the hepatopancreas of red claw crayfish in the VM and HM groups were significantly lower than those in the control group (P<0.05).

[0099] As shown in Table 4, only the proportion of lysine in the ovaries of red claw crayfish in the BM group was significantly lower than that in the control group (P<0.05), while the proportions of other amino acids were not significantly different from those in the control group (P>0.05). The proportions of aspartic acid, glutamic acid, methionine, isoleucine, tyrosine, histidine and lysine in the ovaries of the HM group were significantly lower than those in the control group (P<0.05), and the proportions of other amino acids were not significantly different from those in the control group (P>0.05). The proportion of valine in the ovaries of the TM group was not significantly different from that in the control group (P>0.05), and the proportions of other amino acids were significantly lower than those in the control group (P<0.05). The proportions of each amino acid in the ovaries of red claw crayfish in the VM group were significantly lower than those in the control group (P<0.05).

[0100] Protein content and amino acid composition can significantly affect the gonadal development of crustaceans, and appropriate feed amino acid nutrition can effectively improve the survival, growth, development, molting rate and reproduction of crustaceans. In this embodiment, compared with the control group, the amino acid composition of the hepatopancreas and ovaries of red claw crayfish in the BM group was most similar to that of the control group, and the amino acid ratio of the hepatopancreas and ovaries of red claw crayfish in the VM group, TM group and HM group was slightly lower overall, indicating that the addition of defatted silkworm pupa powder in the feed can promote the accumulation of protein nutrition in the hepatopancreas and ovaries, which is beneficial to the ovarian development of female broodstock.

[0101] Serum metabolomics analysis

[0102] The sera of red claw crayfish in the FM group and the BM group were selected for differential metabolite analysis (n=6), which included the following steps:

[0103] Add 400 μL of isotope-labeled extract (methanol: acetonitrile = 1:1 (V / V)) to 100 μL of serum, mix well, ultrasonicate for 10 min, and stand at -40°C for 1 h. Centrifuge (12000 rpm, 4°C) for 15 min, take the supernatant for detection, and take an equal amount of supernatant from all samples and mix them as quality control (QC) samples;

[0104] An ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher) and a liquid chromatography column (ACQUITY UPLC BEH Amide, 2.1 mm × 100 mm, 1.7 μm, Waters Corporation) were used for chromatographic separation, and a mass spectrometer (Thermo Q Exactive HFX, Thermo Fisher) was used for mass spectrometry data acquisition. The data were processed by ProteoWizard software, and substance annotation was performed. Finally, differential metabolites were screened, visualized, and further bioinformatics analysis was performed.

[0105] When metabolomics analysis was performed on the serum of red claw crayfish in the FM and BM groups, a total of 8815 Peaks were extracted. After pretreatment, 8597 Peaks were retained and matched with the mass spectrometry database for material annotation. All metabolites were divided into 14 categories, among which the largest ones were lipids and lipid molecules (37.374%), organic acids and their derivatives (21.212%), etc. ( Figure 7 In the results of principal component analysis (PCA), it can be found that the experimental samples are all within the 95% confidence interval ( Figure 7In the OPLA-DA model score scatter plot, there were differences in metabolites between the BM and FM groups ( Figure 7 In the OPLS-DA permutation test, the corresponding OPLS-DA model was established multiple times (n=200) to obtain the R2 and Q2 values ​​of the random model, where R2Y=0.996 and Q2=0.487, that is, the fitting accuracy of the established model is consistent with the sample reflection situation ( Figure 7 The volcano plot was used to analyze the up- and down-regulation of differential metabolites in the BM group compared with the control group. There were 769 up-regulated differential metabolites and 768 down-regulated differential metabolites ( Figure 7 E in the .

[0106] KEGG enrichment analysis was performed on the differential metabolites. The differential metabolites were mainly enriched in amino acid metabolism and fatty acid metabolism. The amino acid metabolism pathways were tryptophan metabolism, arginine and proline metabolism, lysine degradation, and arginine biosynthesis. The fatty acid metabolism pathways were alpha-linolenic acid metabolism, linoleic acid metabolism, and glycerophospholipid metabolism. Figure 8 ). According to the enrichment results of differential metabolites in KEGG metabolic pathways, the differential metabolites in metabolic pathways such as arachidonic acid metabolism, linoleic acid metabolism, 2-oxocarboxylic acid metabolism, amino acid biosynthesis metabolism and lysine degradation were significantly enriched ( Fig. 9 , P<0.05).

[0107] Comprehensive analysis of the differential metabolite pathways (enrichment analysis and topological analysis) revealed that linoleic acid metabolism, D-glutamine and D-glutamate metabolism, alanine, aspartate and glutamate metabolism, tryptophan metabolism, arginine and proline metabolism were the key pathways with high correlation of metabolite differences ( Fig.10 ), thereby one or more of linoleic acid, D-glutamine, D-glutamate, alanine, aspartic acid, glutamic acid, tryptophan, arginine and proline can be used as differential metabolites to detect the ovarian development of female shrimp.

[0108] Amino acids are used to synthesize proteins in the body and serve as precursors for many molecules, including neurotransmitters, hormones, purines, pyrimidines, and vitamins. Amino acid degradation is one of the important ways for organisms to use amino acids to generate energy. Among them, lysine is an essential amino acid for crustaceans. Although crustaceans lack the biosynthetic machinery required to synthesize lysine from the source, they can decompose lysine and use it to synthesize biological molecules in the physiological metabolism of the body. In this experiment, the metabolic pathway of lysine degradation in red claw crayfish in the BM group showed an up-regulated trend, which improved the metabolism of the red claw crayfish.

[0109] At the same time, tryptophan is the third most limiting amino acid in low-protein feeds after lysine and methionine. In invertebrates, the neurotransmitter serotonin (5-HT) can regulate the maturation of the ovaries, and tryptophan, as a precursor of 5-HT, is a prerequisite for the synthesis of 5-HT. In this experiment, the tryptophan metabolic pathway in the serum of red claw crayfish in the BM group showed an up-regulated trend, which indicates that it can play a positive role in promoting the maturation of the ovaries of red claw crayfish.

[0110] In this embodiment, the expression trend of differential metabolites in the glycerophospholipid metabolic pathway is upregulated. Glycerophospholipids are involved in a variety of metabolic and signal transduction functions. Due to their hydrophilicity and hydrophobicity, they are essential for maintaining the double-layer structure and homeostasis of cell membranes and achieving vascular transport of lipids. The results of the serum metabolome showed that the addition of defatted insect protein sources (especially defatted silkworm pupa powder) to the red claw crayfish parent feed can increase the enrichment of amino acid and fatty acid related metabolites, that is, when the ovaries develop, the nutrients in the hemolymph of crayfish that ingest defatted silkworm pupa powder are more enriched, and the substances transported to the ovaries will also increase accordingly, which is beneficial to the accumulation of nutrients in the ovaries.

[0111] In summary, since the three defatted insect protein sources in the present invention belong to the same phylum as shrimp, and have high protein content, relatively balanced amino acid composition, low fat content that is easily oxidized and rancid, and are rich in bioactive substances such as vitamins, minerals, and antimicrobial peptides unique to insect protein that are beneficial to the regulation of body health, they can significantly improve the growth performance of aquatic animals and enhance the immunity of aquatic animals. However, there are few reports on the research of defatted insect protein sources in female broodstock feed.

[0112] Therefore, the present invention can promote the ovarian development of female broodstock to different degrees by adding three defatted insect protein sources to the female broodstock feed, especially after adding defatted silkworm pupa powder, which can be used as the main protein source, significantly improve the sex hormone level of female broodstock serum, and increase the metabolite enrichment of amino acid and fatty acid related metabolic pathways such as lysine degradation, amino acid biosynthesis, glycerophospholipids, arachidonic acid and 2-oxocarboxylic acid metabolic pathways, effectively increase the protein content of the hepatopancreas and the accumulation of triglycerides in the ovary, promote the development of the ovary, and improve the quality of ovarian development.

[0113] The technical features in the above embodiments can be combined arbitrarily, and the combined technical solutions all belong to the protection scope of this application. The basic principles, main features and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may have various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements all fall within the scope of the present invention to be protected. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of a defatted insect protein source in preparing a feed for promoting ovarian development of female broodstock.

2. The use according to claim 1, characterized in that The defatted insect protein source includes one or more of defatted yellow mealworm powder, defatted black soldier fly powder and defatted silkworm pupa powder.

3. The use according to claim 2, characterized in that By weight, the crude protein content of the defatted mealworm powder is 60-70%, and the crude fat content is 8-10%, the crude protein content of the defatted black soldier fly powder is 45-55%, and the crude fat content is 10-15%, and the crude protein content of the defatted silkworm pupa powder is 50-60%, and the crude fat content is 10-18%.

4. A feed for promoting ovarian development of female broodstock, characterized in that: By weight, each kilogram of the feed includes: 100-200g of fish meal, 150-200g of defatted insect protein source, 145-180g of microcrystalline cellulose, 150-200g of gluten, 50-80g of soybean oil, 0.3-1.0g of 2,6-di-tert-butyl-4-methylphenol, 5-15g of krill oil, 150-200g of wheat starch, 4-6g of cholesterol, 15-25g of vitamin premix, 15-25g of mineral premix, 1-5g of choline chloride, 15-25g of sodium carboxymethyl cellulose, 5-15g of monocalcium phosphate, and 0.3-1.0g of cuttlefish paste.

5. The feed according to claim 4, characterized in that The feed does not include soybean meal, cottonseed meal and peanut meal.

6. Use of the feed according to any one of claims 4 to 5 for increasing the nutrient content of the hepatopancreas and / or ovary of female broodstock.

7. The use according to claim 6, characterized in that The hepatopancreas nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose, and the ovarian nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose.

8. Use of the feed according to any one of claims 4 to 5 in increasing the content of sex hormones in the serum of female broodstock.

9. The use according to claim 8, characterized in that The sex hormones include one or both of estradiol and progesterone.

10. Use of differential metabolites produced after feeding the feed according to any one of claims 4 to 5 to female broodstock in detecting ovarian development of female broodstock.

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