Application of degreased silkworm chrysalis meal in promoting ovarian development of female parent shrimps

Abstract

The invention discloses an application of defatted silkworm chrysalis meal in promoting ovarian development of female parent shrimps, after different proportions of defatted silkworm chrysalis meal is added into a feed, the serum sex hormone level of the female parent shrimps can be improved, the accumulation of nutrient substances of hepatopancreas, ovaries and fertilized eggs is increased, and the ovarian development is effectively promoted; the proportion of oviposition female shrimps is increased, the weight of fertilized eggs is increased, the reproductive performance is effectively improved, and scientific reference is provided for improving the gonad development quality of female parent shrimp individuals.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic feed, and in particular to application of defatted silkworm pupa (Bombyx mori) powder in promoting ovarian development of female broodstock. Background Art

[0002] Artificial feed is the most stable source of nutrition for broodstock and is crucial to the growth and development of broodstock. At present, the nutritional research on the maturation of female broodstock of red claw crayfish (Cherax quadriarinatus) is limited and relatively backward, and there is no special feed for red claw crayfish broodstock on the market.

[0003] During the stage of nutrition enhancement for red claw crayfish, compound feeds of shrimps such as Penaeus vannamei and Macrobrachium rosenbergii are often used for feeding, but they do not fully meet the nutritional requirements of omnivorous red claw crayfish during the stage of nutrition enhancement, so they need to be supplemented with fresh animal protein sources and plant protein sources. However, fresh animal protein sources are not only difficult to store, but also difficult to control the source and quality, and are very easy to pollute the water quality and even carry pathogens. There is uncertainty about the impact on the ovarian development of red claw crayfish, and it is impossible to provide seedlings in large quantities and stably, and it is impossible to meet the needs of large-scale farming of red claw crayfish. Summary of the invention

[0004] The invention provides a use of defatted silkworm pupa (Bombyx mori) powder in promoting ovarian development of female broodstock, aiming to solve the problems existing in the above-mentioned background technology.

[0005] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0006] On the one hand, a use of defatted silkworm pupa (Bombyx mori) powder in preparing a feed for promoting ovarian development of female broodstock is provided.

[0007] Preferably, the female broodstock comprises red claw crayfish (Cherax quadriarinatus).

[0008] Preferably, the defatted silkworm pupa powder has a crude protein content of 50-60% and a crude fat content of 10-18% by weight.

[0009] On the other hand, a feed for promoting the ovarian development of female broodstock is provided. The feed comprises 9.0% to 37.0% by weight of defatted silkworm pupa powder per kilogram.

[0010] Preferably, per kilogram of the feed comprises: 0-300g of fish meal, 90-370g of defatted silkworm pupa powder, 150-200g of wheat starch, 150-200g of gluten, 110-125g of microcrystalline cellulose, 0-20g of fish oil, 30-50g of soybean oil, 5-15g of krill oil, 4-6g of cholesterol, 0.3-1.0g of 2,6-di-tert-butyl-4-methylphenol, 0.3-1.0g of cuttlefish paste, 8-12g of vitamin premix, 15-25g of mineral premix, 10-20g of monocalcium phosphate, 5-15g of choline chloride, and 15-25g of sodium carboxymethyl cellulose, and the feed does not include soybean meal, cottonseed meal and peanut meal.

[0011] Preferably, per kilogram of the feed comprises: 0-300g of fish meal, 180-280g of defatted silkworm pupa powder, 150-200g of wheat starch, 150-200g of gluten, 110-125g of microcrystalline cellulose, 0-20g of fish oil, 30-50g of soybean oil, 5-15g of krill oil, 4-6g of cholesterol, 0.3-1.0g of 2,6-di-tert-butyl-4-methylphenol, 0.3-1.0g of cuttlefish paste, 8-12g of vitamin premix, 15-25g of mineral premix, 10-20g of monocalcium phosphate, 5-15g of choline chloride, and 15-25g of sodium carboxymethyl cellulose, and the feed does not include soybean meal, cottonseed meal and peanut meal.

[0012] Furthermore, every 100 g of the vitamin premix includes the following raw materials in parts by weight:

[0013] 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.

[0014] Furthermore, every 100 g of the mineral premix includes the following raw materials in parts by weight:

[0015] 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.

[0016] 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 and / or fertilized eggs of female broodstock.

[0017] Preferably, the hepatopancreas nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose, the ovarian nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose, and the fertilized egg nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose.

[0018] 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.

[0019] Preferably, the sex hormone includes one or both of estradiol and progesterone.

[0020] 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.

[0021] Preferably, the differential metabolites include one or more of alanine, aspartic acid, and glutamate.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] After adding defatted silkworm pupa powder in different proportions to the feed of the present invention, the serum sex hormone level of female broodstock can be improved, the accumulation of nutrients in the hepatopancreas, ovaries and fertilized eggs can be increased, the development and maturation of ovaries can be effectively promoted, the proportion of spawning female shrimps and the weight of fertilized eggs can be increased, and the reproductive performance can be effectively improved, providing a scientific reference for improving the gonad development quality of female broodstock individuals. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 Effects of defatted silkworm pupa powder on the growth performance of female red claw crayfish: (A) survival rate; (B) weight gain rate; (C) fatness; (D) liver-to-body ratio; (E) gonad index;

[0026] Figure 2 Effects of defatted silkworm pupa powder on the reproductive performance and embryo quality of female red claw crayfish, (A) Proportion of spawning females; (B) Weight of fertilized eggs; (C) Absolute egg production; (D) Relative egg production; (E) Weight of juvenile shrimp;

[0027] Figure 3 The developmental stages of the female red claw crayfish ovary after H&E staining, C: ooplasm; Oo: oocyte; Y: yolk granules; N: nucleus; PVS: perivitelline space;

[0028] Figure 4 The effect of defatted silkworm pupa powder on the content of nutrients in the hepatopancreas of female red claw crayfish: (A) total protein; (B) triglyceride; (C) total cholesterol; (D) glucose; (E) glycogen;

[0029] Figure 5 The effect of defatted silkworm pupa powder on the nutrient content of female red claw crayfish ovary, (A) total protein; (B) triglyceride; (C) total cholesterol; (D) glucose; (E) glycogen;

[0030] Figure 6 The effect of defatted silkworm pupa powder on the nutrient content of fertilized eggs produced by female red claw crayfish. (A) Total protein; (B) Triglyceride; (C) Total cholesterol; (D) Glucose; (E) Glycogen;

[0031] Figure 7 The effect of defatted silkworm pupa powder on the serum sex hormone levels of female red claw crayfish: (A) estradiol; (B) progesterone; (C) methyl farnesate;

[0032] Figure 8 The statistical diagram of differentially expressed genes between the 18.4% group and the control group, (A) statistical diagram of differentially expressed genes: red and blue represent the total number of genes that are significantly up-regulated and significantly down-regulated, respectively; (B) volcano diagram of differentially expressed genes: red, blue and gray represent genes that are significantly up-regulated, significantly down-regulated and have no significant difference, respectively;

[0033] Fig. 9 The GO enrichment analysis results of differentially expressed genes between the 18.4% group and the control group. The larger the RichFactor, the higher the degree of enrichment. GO Term is the GO functional annotation. The size and color of the bubble represent the number of differentially expressed genes and the Q value (corrected value of P.value, representing the significance of enrichment).

[0034] Fig.10 The biological processes of differentially enriched genes between the 18.4% group and the control group, with the vertical and horizontal axes representing GO terms and -log10(Q) values ​​of GO term enrichment, respectively;

[0035] Fig.11 The molecular functions of differentially enriched genes between the 18.4% group and the control group, the vertical axis and horizontal axis are GO terms and -log10(Q) values ​​of GO term enrichment, respectively;

[0036] Fig.12This is the KEGG enrichment analysis diagram of differentially expressed genes between the 18.4% group and the control group. The horizontal and vertical axes represent the number of differentially expressed genes and the name of the pathway, respectively.

[0037] Fig.13 This is the KEGG enrichment bubble chart of the differentially expressed genes between the 18.4% group and the control group. The larger the enrichment factor on the horizontal axis, the higher the enrichment degree of the pathway. The vertical axis is the KEGG pathway. The size and color of the bubbles represent the number of differentially expressed genes and the P value of the enrichment analysis, respectively. DETAILED DESCRIPTION

[0038] 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.

[0039] Example:

[0040] Experimental design and preparation of experimental feed

[0041] 1. Nutritional fortification experiment

[0042] The female and male broodstock used in this experiment were red claw crayfish purchased from the Guangxi Agricultural Seed Hainan South Breeding Base in Ledong Li Autonomous County, Hainan Province, China.

[0043] In this experiment, a total of 5 treatment groups were set up, including 0% group (control group), 9.2% group, 18.4% group, 27.6% group and 36.8% group, each treatment group had 4 parallels, and each parallel had 10 female broodstock.

[0044] Red claw crayfish need to be temporarily reared for 5 days to adapt to the experimental environment. During the temporary rearing period, they are fed with commercial pellet feed (such as Tongwei feed, etc.) twice a day. Six-month-old female broodstock (40.00±2.00g) with consistent specifications, health and complete appendages are randomly selected and randomly distributed to baskets (length×width×height=25.8cm×19.5cm×15.8cm) with foam bars fixed with tie bands. One female broodstock is placed in each basket to avoid crayfish killing each other during the experiment, thereby affecting the accuracy of the experiment. During the nutritional fortification experiment, 10 frames are also set for each parallel, and one female broodstock of each group is placed in each frame for breeding.

[0045] At the same time, this experiment set up 5 groups of equal nitrogen and equal fat feeds (i.e., "experimental feeds" in Table 1), the feed ingredients and formulas are shown in Table 1, during the nutritional fortification experiment, the 0% group, 9.2% group, 18.4% group, 27.6% group and 36.8% group were fed with experimental feeds with mass fractions of defatted silkworm pupa powder of 0%, 9.2%, 18.4%, 27.6% and 36.8%, respectively, and the nutritional fortification experiment time was 50 days. It should be noted that in conventional shrimp farming basic feed, the mass fraction of fish meal as the main protein source is usually about 36.8%, so in this embodiment, the mass fraction gradient of defatted silkworm pupa powder is set according to this ratio (i.e., 36.8%).

[0046] Table 1 Formulas of five experimental feeds (g / kg dry weight) and approximate composition (% dry weight)

[0047]

[0048]

[0049] Among them, defatted silkworm pupa powder was purchased from Haikun Agricultural Technology Co., Ltd.

[0050] 2. Mating experiment

[0051] In this experiment, a total of 5 treatment groups were set up (corresponding to the 0% group, 9.2% group, 18.4% group, 27.6% group and 36.8% group). After 50 days of nutritional fortification experiment, 24 female shrimps were selected from each of the 0% group, 9.2% group, 18.4% group, 27.6% group and 36.8% group of the nutritional fortification experiment for mating experiments.

[0052] Among them, each treatment group of the mating experiment was mated in a cement pool (length × width × height = 4.0m × 1.5m × 0.9m). First, 10 male red claw crayfish were placed in a cement pool with two-thirds of water and shelter. After 5 days of temporary cultivation, 24 female red claw crayfish selected from the treatment group of the same nutrition fortification experiment were placed for mating experiment. The mating experiment lasted for 28 days. After the mating experiment, the female shrimps were incubated with eggs for 47 days, and the hatched shrimps in each treatment group of the mating experiment were weighed.

[0053] The male and female shrimps in each treatment group were raised at the same time for mating experiments. Before the mating experiment, they were fed with commercial pellet feed (such as Tongwei feed, etc.) twice a day, and male red claw crayfish (60.00±2.00g) of uniform size, health, strong vitality and complete appendages were selected for mating.

[0054] 3. Experiment on acute ammonia nitrogen stress in juvenile shrimp

[0055] In this experiment, a total of 5 treatment groups were set up (corresponding to the 0% group, 9.2% group, 18.4% group, 27.6% group and 36.8% group), with 3 parallels in each group. Fifty juvenile shrimps (0.072-0.180 g) produced by the broodstock of the treatment group in the same mating experiment were selected from each parallel of each group and placed in a transparent polypropylene plastic box (length × width × height = 36.5 cm × 25.0 cm × 11.3 cm) for a 96-h acute ammonia nitrogen stress experiment. During the experiment, the ammonia nitrogen concentration in the water was detected with an ammonia nitrogen analyzer (PT-001B, Pantian Biotech, China), and the ammonia nitrogen concentration in the breeding water of each group was 12.5 mg / L by replacing the water, and the number of surviving juvenile shrimps in each group was counted at 0 h, 24 h, 48 h, 72 h and 96 h, respectively.

[0056] Farming management and experimental conditions

[0057] During the nutritional fortification experiment, each group was fed the corresponding experimental feed in a fixed amount and at regular intervals every day. In each treatment group, the daily feed intake was 4% of the body weight of the red claw crayfish, and it was fed at 8:00 and 16:00 every day. Remove residual feed and feces 2 hours after feeding. In addition, in each treatment group, two-thirds of the water was replaced every three days to maintain the best water quality. During the breeding period, the average temperature of the breeding 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.

[0058] During the mating experiment, each treatment group was fed with the corresponding experimental feed at 8:00 and 16:00 every day, and the water was changed once a week. Each time the water was changed, it was enough for the red claw crayfish to swim, so as to observe and count the female shrimps carrying eggs. During the hatching stage after the mating experiment, the experimental feed was fed at 8:00 and 16:00 every day, and one-third of the water was changed every three days until each group of fertilized eggs was successfully hatched. During the breeding period, the ammonia nitrogen in the water of each group of breeding water was <0.05mg / L, the average temperature was 28.0±2.0℃, the average pH was 7.60±0.20, and the dissolved oxygen was ≥5.0mg / L.

[0059] During the acute ammonia nitrogen stress experiment of juvenile shrimp, water was changed in each treatment group at 0h, 24h, 48h, 72h and 96h, and all water bodies were replaced and continuously monitored with an ammonia nitrogen analyzer to maintain the ammonia nitrogen concentration of the water at 12.5mg / L. The other water parameters were the same as those in the mating experiment.

[0060] Sample collection

[0061] Before the end of the nutritional fortification experiment, the female shrimps in each group were fasted for 24 hours. After the nutritional fortification experiment, female shrimps were selected from each group for ice bath anesthesia, and the final weight was measured after the surface moisture of the female shrimps was wiped dry. Hemolymph was extracted from the pericardial cavity of the female shrimps 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 samples for the detection of sex hormone content.

[0062] The carapace was cut with ophthalmic scissors, and the ovarian and hepatopancreatic tissues were quickly separated with forceps. The hepatopancreas and ovaries of 4 female shrimps 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.

[0063] During the mating experiment, the fertilization and egg-carrying status of female shrimps were observed when the water was changed every week. Female shrimps with eggs were collected from each group and weighed. The female shrimps and eggs were carefully separated with a comb. The number of shrimp eggs in each group was then counted and weighed, and placed in 2.0 mL cryovials and stored in a -80°C refrigerator.

[0064] The juvenile shrimps used in the acute ammonia nitrogen stress experiment were collected using a honeycomb plastic shelter, and the collected juvenile shrimps were temporarily raised in a plastic aquarium (length×width×height=54.2cm×42.0cm×37.0cm) for the acute ammonia nitrogen stress experiment.

[0065] Phenotypic parameter statistics

[0066] The corresponding phenotypic parameters were calculated according to the following formulas:

[0067] Weight gain (WG, %) = 100% × [final body weight (g) - initial body weight (g)] / initial body weight (g)

[0068] Condition factor (CF, %) = 100% × final body weight (g) / [body length (cm)] 3

[0069] Survival rate (%) = 100% × final number / initial number

[0070] Hepatosomatic index (HSI, %) = 100% × hepatopancreas weight (g) / body weight (g)

[0071] Gonadal index (GSI, %) = 100% × ovarian weight (g) / body weight (g)

[0072] Percentage of female spawned (%) = 100% × number of females with eggs / number of surviving females Absolute egg production (grain / stern) = total number of eggs laid by each female

[0073] Relative egg production (grain / g) = total number of eggs laid by each female shrimp / female shrimp weight

[0074] like Figure 1 As shown in the figure, the survival rate of female shrimp in each group of nutritional fortification experiment ( Figure 1 Middle A part), fatness ( Figure 1 C), liver-to-body ratio ( Figure 1 D section) and gonadal index ( Figure 1 There was no significant difference in the weight gain of female shrimps (P>0.05). However, when the mass fraction of defatted silkworm pupa powder in the feed exceeded 9.2%, the weight gain rate showed a downward trend, and the weight gain rate of female shrimps in the 9.2% group was significantly higher than that in the 27.6% and 36.8% groups ( Figure 1 Part B, P < 0.05).

[0075] like Figure 2 As shown in the results, during the mating experiment, at the 14th and 28th days of mating, the proportion of spawning female shrimp increased with the increase of the proportion of defatted silkworm pupa powder in the feed. At the 14th day, the proportion of spawning female shrimp in the 18.4%, 27.6% and 36.8% groups was significantly higher than that in the 9.2% and control groups (P<0.05); at the 28th day, the proportion of spawning female shrimp in the 27.6% and 36.8% groups was significantly higher than that in the control group ( Figure 2 The weight of fertilized eggs produced by female shrimps in the 18.4% and 27.6% groups was significantly higher than that in the 36.8% group ( Figure 2 The absolute and relative spawning amount of crayfish in the 9.2% group was significantly higher than that in the 18.4% group ( Figure 2 In the C and D parts of the incubation, P<0.05). In the results of the weight of the juvenile shrimps at 47 days of incubation, the weight of the juvenile shrimps in the 36.8% group was the heaviest (P<0.05), followed by the 27.6% group (P<0.05). There was no significant difference in the weight of the juvenile shrimps between the 18.4% group and the control group (P>0.05), but the weight of the juvenile shrimps in the 9.2% group was significantly lower than that in the control group ( Figure 2 (Part E, P < 0.05).

[0076] In this embodiment, the weight of fertilized eggs showed a trend of first increasing and then decreasing with the increase of the proportion of defatted silkworm pupa powder in the feed, among which the fertilized eggs in the 18.4% group were the heaviest, but since the red claw crayfish only laid 100 to 1000 eggs each time, its absolute and relative egg laying amount would also be relatively reduced. In addition, the triglyceride and total cholesterol contents of the fertilized eggs in the 18.4% group were both high, indicating that adding 18.4% defatted silkworm pupa powder to the feed can increase the nutrient content of the fertilized eggs.

[0077] At the same time, as shown in Table 2, in the acute ammonia nitrogen stress experiment on juvenile shrimp, under the conditions of ammonia nitrogen stress, the survival rate of juvenile shrimp in the 36.8% group at 48h, 72h and 96h was the highest among all groups (P<0.05), followed by the 18.4% group, while the survival rate of juvenile shrimp in the 9.2% group at 24h, 48h, 72h and 96h was relatively low (Table 3.5, P>0.05).

[0078] Table 2 Effects of acute ammonia nitrogen stress on the survival rate of juvenile shrimp

[0079]

[0080] Proper nutrition intake is crucial for the reproductive performance of aquatic animal parents, and the nutritional level of feed significantly affects the maturation, reproductive capacity, embryonic development, larval quality and larval survival rate of the parents. In this embodiment, the proportion of female spawning shrimps on the 14th and 28th days of mating increased with the increase of the proportion of defatted silkworm pupa powder in the feed.

[0081] At the same time, in the acute ammonia nitrogen stress experiment on juvenile shrimp, the survival rate of the juvenile shrimp was affected to a certain extent due to the difference in body weight, but the survival rates of the juvenile shrimp in the 18.4% group, 27.6% group and 36.8% group at different time points were mostly higher than those of the control group. This also shows to a certain extent that adding defatted silkworm pupa powder to the feed can promote the development of juvenile shrimp and improve its tolerance to acute ammonia nitrogen stress.

[0082] Histological observation

[0083] After the nutritional fortification experiment, the ovarian tissues of each group of broodstock obtained by sampling were uniformly selected from the upper left half of the ovary for making tissue sections, and the hematoxylin-eosin (H&E) staining method was used, which included the following steps:

[0084] The ovarian tissue samples were fixed in a 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.

[0085] The ovarian tissue samples were sliced ​​with a slicer (thickness of about 5 μm) and stored at -20°C for later use;

[0086] 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).

[0087] like Figure 3 As shown in the figure, the ovaries of female shrimps in the 18.4%, 27.6% and 36.8% groups were mostly in the middle or late stages of development. As the oocytes increased in volume, they squeezed each other, showing polygonal or elliptical structural characteristics, and the nuclei gradually disappeared. The ovaries of red claw crayfish in the 0% and 9.2% groups were in the early or middle stages of development, the oocytes developed asynchronously and the perivitelline space was larger. This shows that the addition of defatted silkworm pupa powder in the feed is positively correlated with the development and maturation of the female shrimp ovaries. When the addition ratio of defatted silkworm pupa powder is greater than or equal to 18.4%, it can promote ovarian development.

[0088] Determination of nutrients and sex hormone content

[0089] In the nutrition fortification experiment, two hepatopancreas tissue samples and two ovarian tissue samples were randomly taken from each parallel group; in the mating experiment, one fertilized egg was taken from each group;

[0090] 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.

[0091] The processing method of ovarian tissue samples and fertilized eggs is the same as that of hepatopancreatic tissue samples mentioned above, and the total protein, triglyceride, total cholesterol, glycogen and glucose content are also tested.

[0092] The above indicators of hepatopancreas, ovary and fertilized egg 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).

[0093] In each parallel of each group of the nutritional fortification experiment, two serum samples were randomly taken and tested for sex hormone content, wherein the sex hormone content includes one or more of estradiol, progesterone and methyl farnesoate.

[0094] 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.

[0095] like Figure 4 As shown in the figure, in each group of the nutritional fortification experiment, the triglyceride content in the hepatopancreas of female shrimp ( Figure 4 B), total cholesterol ( Figure 4 C part) and glucose ( Figure 3 There was no significant difference in the content of D in the feed (P>0.05). However, the total protein content of the 9.2%, 18.4%, 27.6% and 36.8% groups was higher than that of the control group. When the amount of defatted silkworm pupa powder added to the feed reached 18.4%, the total protein content of the hepatopancreas of female shrimp was the highest (P>0.05), and was significantly higher than that of the control group ( Figure 4 The glycogen content of the hepatopancreas of female shrimp in the 9.2% group, 18.4% group and control group was significantly higher than that in the 27.6% group ( Figure 4 (Part E, P < 0.05).

[0096] like Figure 5 As shown in the figure, in each group of the nutritional fortification experiment, the total protein in the female shrimp ovaries ( Figure 5 A in the Figure 4 There was no significant difference in the content of triglyceride in the E part of the ovaries of female shrimps in the 9.2% group, 18.4% group and control group (P>0.05). The triglyceride content in the ovaries of female shrimps in the 9.2% group, 18.4% group and control group was significantly higher than that in the 27.6% group and 36.8% group ( Figure 5 The total cholesterol levels in the 9.2%, 18.4%, 27.6% and 36.8% groups were higher than those in the control group ( Figure 5 Meanwhile, the glucose content in the ovaries of female shrimps in the 9.2%, 18.4% and 27.6% groups was significantly higher than that in the 36.8% and control groups ( Figure 5 (Part D, P < 0.05).

[0097] like Figure 6 As shown, the total protein in the fertilized eggs produced by female shrimps in each group ( Figure 6 A part of the Figure 6D part) and glycogen ( Figure 6 There was no significant difference in the content of triglyceride in the E part of the 18.4% group (P>0.05). The triglyceride content in the fertilized eggs produced by female shrimps in the 18.4% group was significantly higher than that in the other treatment groups ( Figure 6 The total cholesterol content in the fertilized eggs of crayfish in the 9.2% group was significantly higher than that in the 27.6% group ( Figure 6 (Part C, P < 0.05).

[0098] The nutrient accumulation in the ovary and hepatopancreas is a key indicator of the reproductive biology of crustaceans. The nutrient content of the hepatopancreas of crustacean pre-adults will affect the accumulation of nutrients in the ovary, and ultimately affect indicators such as egg quality, hatching rate and survival rate of larvae, among which protein is the main nutrient required for ovarian development. In this embodiment, the total protein content of the hepatopancreas of female shrimps showed a trend of first increasing and then decreasing with the proportion of defatted silkworm pupa powder in the feed, but the total protein content of the hepatopancreas of female shrimps in the 9.2% group, the 18.4% group, the 27.6% group, and the 36.8% group was higher than that in the control group, and the addition of defatted silkworm pupa powder did not have a negative impact on the total protein content of the ovaries, indicating that the addition of defatted silkworm pupa powder in the feed can increase the total protein content of the hepatopancreas of female shrimps to promote the ovarian development of female shrimps.

[0099] The lipid levels of the hepatopancreas and ovaries of crustaceans play an important role in the metabolism and reproduction of crustaceans, especially neutral lipids such as triglycerides, which are the main storage form and source of energy required by crustacean adults, eggs and yolk-nutritional larvae. In this example, the triglyceride content of the ovaries of female shrimps in the 0%, 9.2% and 18.4% groups was significantly higher than that in the 27.6% and 36.8% groups, and the triglyceride content in the fertilized eggs produced by female shrimps in the 18.4% group was the highest among all treatment groups. In the transcriptomic GO enrichment analysis of the 18.4% group versus the control group described below, the differentially expressed genes were enriched in glycerol channel activity, which can improve the transport capacity of triglycerides, indicating that the addition of 18.4% defatted silkworm pupa powder to the feed is beneficial to the accumulation of triglycerides in crayfish ovaries.

[0100] In addition, cholesterol is an important sterol and a precursor of many physiologically active compounds, such as sex hormones, ecdysone, adrenocortical hormones, bile acid and vitamin D. Cholesterol is also often used as an important indicator in the evaluation of the nutritional status of crustacean parents. In this example, there was no significant difference in the total cholesterol content in the hepatopancreas and ovaries of female shrimps in each group, indicating that adding defatted silkworm pupa powder to the feed would not have a negative impact.

[0101] In addition, carbohydrates in feed are an important component to meet the energy needs of farmed animals, and the intake of carbohydrates is conducive to the storage of glycogen. In this embodiment, when the proportion of defatted silkworm pupa powder added to the feed is greater than 18.4%, the glycogen content of the hepatopancreas of crawfish shows a downward trend. The reason is that, as mentioned above, the amount of defatted silkworm pupa powder added to the feed is positively correlated with the development and maturation of the crawfish ovaries. It is speculated that it may be due to the improvement of ovarian maturity, the biosynthesis and nutrient mobilization during ovarian development consume more energy substances, and the glycogen of the hepatopancreas is the energy source for maintaining life activities, so the glycogen content of the hepatopancreas of female shrimps shows a downward trend. In addition, the glucose content of the female shrimp ovaries in the 9.2% group, the 18.4% group and the 27.6% group is relatively high, indicating that defatted silkworm pupa powder can promote the increase of glucose content in the female shrimp ovaries, and further provide energy through glucose for biosynthesis.

[0102] At the same time, in the transcriptomic GO enrichment analysis results of the 18.4% group compared with the control group, it can be seen that the differential genes are enriched in trehalose transport and trehalose transmembrane transporter activity, and the glucose content in the ovaries of female shrimps in the 18.4% group is significantly higher than that in the control group, thereby improving the carbohydrate transport capacity into the ovaries of female shrimps. At the same time, through KEGG enrichment analysis, the upregulated differential genes are significantly enriched in the pentose and glucuronic acid mutual conversion pathway, indicating that the addition of 18.4% defatted silkworm pupa powder to the feed can significantly improve the metabolic capacity of carbohydrates in the ovaries of female shrimps.

[0103] Furthermore, the triglyceride, total cholesterol, glucose and glycogen contents in the fertilized eggs were the highest in the 9.2% group, indicating that the shrimps fed with feed containing defatted silkworm pupa powder can provide sufficient energy for the development of the larvae, especially the higher cholesterol content in the fertilized eggs may be more conducive to the development of the larvae.

[0104] like Figure 7 As shown in the figure, in each group of the nutritional fortification experiment, the serum estradiol content of female shrimp increased with the increase of the proportion of defatted silkworm pupa powder in the feed, and the serum estradiol content of female shrimp in the 18.4% group was significantly higher than that in the 0% group and the 9.2% group ( Figure 5 The progesterone content in serum showed an opposite trend. The progesterone content in serum decreased with the increase of defatted silkworm pupa powder in feed. The progesterone content in serum of female shrimps in the 18.4% group was significantly higher than that in the 27.6% group and the 36.8% group ( Figure 5 There was no significant difference in the methyl farnesate content in the serum of female shrimps among the groups ( Figure 5C part, P>0.05). This indicates that adding defatted silkworm pupa powder in an appropriate proportion (such as 18.4% by mass) to the feed can promote the increase of estradiol and progesterone levels in the serum of female shrimp, thereby increasing the accumulation of nutrients in female oocytes and promoting the development of the ovaries.

[0105] Analysis of amino acid composition of feed, hepatopancreas and ovarian tissues

[0106] 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.

[0107] 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.

[0108] 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.

[0109] Table 2 Amino acid composition of five experimental feeds (%)

[0110]

[0111]

[0112] 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).

[0113] Table 3 Effect of defatted silkworm pupa powder on amino acid composition of hepatopancreas of female red claw crayfish (% dry weight)

[0114]

[0115] Table 4 Effect of defatted silkworm pupa powder on amino acid composition of hepatopancreas and ovary of female red claw crayfish (% dry weight)

[0116]

[0117]

[0118] As shown in Table 3, the proportion of methionine in the hepatopancreas of female shrimp in the 18.4% group had no significant difference from that in the other treatment groups (P>0.05), and its isoleucine proportion was significantly higher than that in the 27.6% group (P<0.05). The proportions of glutamate, phenylalanine and arginine were the highest among all treatment groups (P<0.05). The proportions of other amino acids had no significant difference from that in the 36.8% group (P>0.05), but were significantly higher than those in the other treatment groups (P<0.05).

[0119] As shown in Table 4, the proportion of methionine in the crayfish ovaries of the 18.4% group had no significant difference from that of the other treatment groups (P>0.05), and its proportions of glutamate, isoleucine and tyrosine were significantly higher than those of the 9.2% group and the 27.6% group (P<0.05), and its proportion of glycine was significantly higher than that of the 27.6% group (P<0.05), and the proportions of serine and arginine were the highest among all treatment groups (P<0.05). The proportions of other amino acids had no significant difference from that of the 36.8% group (P>0.05), but were significantly higher than those of the other treatment groups (P<0.05).

[0120] In the amino acid composition of the feed, except for the lower proportions of tyrosine and phenylalanine in the control group, the proportions of other amino acids were higher. In the amino acid composition of the hepatopancreas and ovary, except for methionine, the 18.4% group had the highest proportion of each amino acid, indicating that female shrimps have a strong ability to utilize the amino acids of defatted silkworm pupa powder, and can further synthesize protein nutrients such as yolk protein through amino acids, improve the synthesis level of yolk protein, hormone peptides and enzymes, and thus promote the maturation and reproduction of the ovaries.

[0121] Ovarian transcriptomic analysis

[0122] In the nutritional fortification experiment, female shrimps from the 18.4% group and the control group were selected for transcriptomics (n=4), which specifically included the following steps:

[0123] The RNA of the female shrimp ovary samples in the 18.4% group and the control group was isolated and purified using Trizol (Thermo Fisher, USA), and the amount and purity of total RNA were detected using NanoDrop ND-1000 (Wilmington, USA), and the integrity of RNA was detected by Bioanalyzer2100 (Agilent, USA) to ensure that the RNA concentration was >50 ng / μL, the RIN value was >7.0, and the total RNA was >1 μg;

[0124] Magnetic beads (Thermo Fisher, USA) were used to capture mRNA with polyadenylation. RNA was fragmented using a magnesium ion shearing kit (NEB Next R Magnesium RNA Fragmentation Module, USA), and cDNA was synthesized using reverse transcriptase (Invitrogen SuperScriptTMII Reverse Transcriptase, USA). E. coli DNA polymerase I (NEB, USA) and RNase H (NEB, USA) were used for double-strand synthesis. At the same time, dUTP Solution (Thermo Fisher, USA) was incorporated into the second strand to fill the ends of the double-stranded DNA into blunt ends, and then an A base was added to each end, and magnetic beads were used for screening and purification;

[0125] The second strand was digested with UDG enzyme (NEB, USA), and then pre-denatured by PCR to form a library with a fragment size of 300±50 bp (strand-specific library). Finally, PE150 double-end sequencing was performed using Illumina NovasekTM 6000.

[0126] like Figure 8 As shown in Figure 2, by comparing the 18.4% group and the control group, a total of 445 differentially expressed genes were screened, of which 290 were up-regulated and 155 were down-regulated ( Figure 8 The differentially expressed genes were visualized and the differences in their expression levels were observed ( Figure 8 in Part B).

[0127] In the Gene Ontology (GO) enrichment analysis of molecular function, cellular component and biological process without distinguishing the differentially expressed genes, the differentially expressed genes were significantly enriched in 18.4% of the group: Trehalose transport, Trehalose transmembrane transporter activity, Proteolysis involved in protein catabolic process, Cysteine-type endopeptidase activity, Glutamate synthase activity, Glycerol channel activity, Acyl-CoA biosynthetic process, Serine-type endopeptidase inhibitor activity and Proteolysis involved in protein catabolic process. Fig. 9 , P<0.05). The hydrolysis of protein is the basis of the reproductive process, including gametogenesis, fertilization and embryonic development. The protein hydrolysis process is the process of breaking down protein into amino acids or small peptide chains, which are the raw materials required for the synthesis of new proteins by organisms. In this embodiment, after adding 18.4% defatted silkworm pupa powder to the feed, the expression of genes related to protein synthesis and decomposition in the ovaries of female shrimps was significantly upregulated, and the upregulated differential genes in the ovaries were significantly enriched in glutamate synthetase activity, which shows that defatted silkworm pupa powder can promote the synthesis and decomposition of proteins, which is beneficial to the synthesis of new proteins.

[0128] In addition, the differentially expressed genes were enriched in the top 20 involved in biological processes, mainly related to the catabolism and transport of proteins, fats and carbohydrates. The differentially expressed genes were significantly enriched in 18.4% of the groups involved in protein catabolism, trehalose transport, acyl-CoA biosynthesis, proteolysis, fatty acid catabolism, triglyceride catabolism, glutamate biosynthetic process, sucrose transport, maltose transport and lactose transport. Fig.10 , P<0.05).

[0129] The differentially expressed genes were enriched in the top 20 molecular functions, which were mainly related to the synthesis, decomposition and transporter activity of amino acids and fats. They were significantly enriched in the 18.4% group for cysteine ​​endopeptidase activity, trehalose transmembrane transporter activity, glutamate synthase activity, glycerol channel activity, serine endopeptidase inhibitor activity, retinoic acid binding, apolipoprotein A-1 binding, N-acetylglucosamine kinase activity, L-aspartate transmembrane transporter activity, Taurine:sodium symporter activity, Serine-type peptidase activity, ionotropic glutamate receptor activity, lipid transporter activity, and lipoprotein transporter activity. Fig.11 , P<0.05).

[0130] Furthermore, in the KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis, the top eight metabolic pathways enriched in differentially expressed genes were amino sugar and nucleotide sugar metabolism, pentose and glucuronate interconversions, purine metabolism, nitrogen metabolism, alanine, aspartate and glutamate metabolism, ascorbate and aldarate metabolism, and porphyrin metabolism. Fig.12 ). The enrichment factor was calculated based on the KEGG enrichment results. The key pathways related to nutrients that were enriched in differentially expressed genes were alanine, aspartate, and glutamate metabolism pathways ( Fig.13 ), thus one or more of alanine, aspartic acid and glutamate can be used as differential metabolites to detect the ovarian development of female shrimp.

[0131] Through the above KEGG enrichment analysis, it was found that the alanine, aspartic acid and glutamate metabolic pathways are key pathways with high correlation of gene differences. This may be due to the fact that the ovaries of female shrimps in the 18.4% group are in a period of rapid development, and yolk protein needs to be continuously synthesized and deposited in the ovaries, and the demand for alanine, aspartic acid and glutamate is large. Secondly, glutamate can be converted into aspartic acid and then regenerated into arginine, and arginine is an essential amino acid for crustaceans, which is beneficial to the molting and reproduction of crustaceans. In this embodiment, the arginine ratio of the 18.4% group feed is low, while the arginine ratio of the corresponding female shrimp ovaries in this group is high, indicating that defatted silkworm pupa powder can improve the gene expression level of alanine, aspartic acid and glutamate metabolic pathways, increase the content of precursor substances such as glutamate used to synthesize arginine in the ovaries, and further improve the protein nutrition level of female shrimps.

[0132] In summary, the crude protein content of defatted silkworm pupa powder is similar to that of fish meal, and the amino acid composition is also relatively balanced, but there are few reports on the research of defatted silkworm pupa powder on female shrimp parents. This application adds defatted silkworm pupa powder in different proportions to the feed, which can improve the serum sex hormone level of female shrimp parents, increase the accumulation of nutrients in the hepatopancreas, ovaries and fertilized eggs, effectively promote the development and maturation of ovaries, increase the proportion of spawning female shrimps and increase the weight of fertilized eggs, and effectively improve the reproductive performance, providing a scientific reference for improving the quality of gonad development of female shrimp parents.

[0133] 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. Application of defatted silkworm pupa powder in preparing feed for promoting ovarian development of female broodstock.

2. The use according to claim 1, characterized in that By weight, the defatted silkworm pupa powder has a crude protein content of 50-60% and a crude fat content of 10-18%.

3. A feed for promoting ovarian development of female broodstock, characterized in that: Measured by weight, each kilogram of the feed contains 9.0%-37.0% by mass of defatted silkworm pupa powder.

4. The feed according to claim 3, characterized in that Each kilogram of the feed includes: 0-300g of fish meal, 90-370g of defatted silkworm pupa powder, 150-200g of wheat starch, 150-200g of gluten, 110-125g of microcrystalline cellulose, 0-20g of fish oil, 30-50g of soybean oil, 5-15g of krill oil, 4-6g of cholesterol, 0.3-1.0g of 2,6-di-tert-butyl-4-methylphenol, 0.3-1.0g of cuttlefish paste, 8-12g of vitamin premix, 15-25g of mineral premix, 10-20g of monocalcium phosphate, 5-15g of choline chloride, and 15-25g of sodium carboxymethyl cellulose.

5. The feed according to claim 3, characterized in that Each kilogram of the feed includes: 0-300g of fish meal, 180-280g of defatted silkworm pupa powder, 150-200g of wheat starch, 150-200g of gluten, 110-125g of microcrystalline cellulose, 0-20g of fish oil, 30-50g of soybean oil, 5-15g of krill oil, 4-6g of cholesterol, 0.3-1.0g of 2,6-di-tert-butyl-4-methylphenol, 0.3-1.0g of squid paste, 8-12g of vitamin premix, 15-25g of mineral premix, 10-20g of monocalcium phosphate, 5-15g of choline chloride, and 15-25g of sodium carboxymethyl cellulose.

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 and / or fertilized eggs 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, the ovarian nutrients include one or more of total protein, triglycerides, total cholesterol, glycogen and glucose, and the fertilized egg 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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