Application of freeze-dried squid powder, feed and shrimp breeding method

Abstract

The invention discloses application of freeze-dried squid powder, feed and a method for breeding shrimps, and relates to the technical field of feed. Wherein the freeze-dried squid powder is used for preparing the feed for promoting gonad development of male shrimps, and in the shrimp feed containing the fish meal, 40-60% of the fish meal is replaced by the freeze-dried squid powder. The invention not only provides a definite evidence for the obvious effect of the squid freeze-dried powder serving as a high-quality feed protein source in promoting the growth and gonad development of male parent shrimps of the penaeus vannamei, but also preliminarily reveals a molecular mechanism for regulating the growth and development of the male shrimps through a specific metabolic pathway; important scientific basis and technical support are provided for efficient and sustainable development of the aquatic product breeding industry.

Description

Technical Field

[0001] The invention relates to the technical field of feed, and in particular to an application of squid freeze-dried powder, feed and a shrimp breeding method. Background Art

[0002] Vannamei shrimp is the shrimp species with the largest farming scale and the largest output in the world. Its healthy farming plays an important role in the sustainable development of aquaculture. The nutritional status of broodstock determines the growth and stress resistance of larvae to a large extent. Therefore, the sustainable development of shrimp farming depends on the healthy development of the broodstock industry to a large extent. In recent years, more and more studies have begun to focus on the nutritional physiology and special feed development of broodstock, but most of the research focuses on female broodstock, while the research on male broodstock is very limited. Male broodstock also need a lot of nutrients during the development of gonads to promote the formation of testes, vas deferens and spermatophore, and to ensure the vitality of sperm. Therefore, the research on the nutritional physiology and feed science of male broodstock should also be put on the agenda as soon as possible to promote the comprehensive and healthy development of shrimp farming.

[0003] In commercial production, the rapid maturation of shrimp gonads is mainly promoted by feeding biological feeds at home and abroad, such as squid, polychaetes and Antarctic krill. The advantages of these biological feeds are that they are rich in protein, fatty acids, cholesterol and other special active substances, especially the high proportion of n-3HUFA in the fatty acid composition of squid and nereid, which is essential for promoting the healthy growth and reproductive capacity of crustaceans. Studies have shown that adding Antarctic krill meal or krill oil to feed can effectively promote the growth of shrimp and Chinese mitten crab and enhance their reproductive performance. In recent years, studies on crustaceans have mostly used the method of feeding biological feeds alone or in combination to explore the effects of different biological feeds on the reproductive performance of shrimp. For example, male pink shrimp (Farfantepenaeuspaulensis) were fed with mixed natural feeds of squid (Illex argentines), blue crabs (Callinectes sapidus) and fish (Macrodon ancylodon) to explore their effects on reproductive performance. In addition, it has been reported that polychaetes can enhance the growth and sperm performance of Penaeus monodon through physiological and morphological analysis. There are also studies that explore the effects of mixing marine bloodworms (Glycera dibranchiata) with other biological baits on the sperm performance of male shrimp. Based on the above studies, biological baits have shown a significant effect in improving the reproductive performance of male broodstock. However, most studies have failed to effectively control the protein and lipid variables of each group of diets under different biological bait treatments, and the evaluation has only stayed at the superficial indicators such as sperm count. There is a lack of in-depth research on the physiological and morphological changes of male shrimps, and detailed reports on how biological baits regulate the metabolic mechanism of male shrimp gonadal development are also relatively lacking. Therefore, it is necessary to conduct in-depth research on the physiological changes and specific mechanisms of gonadal development of male shrimps after ingesting specific biological baits.

[0004] Sperm performance and endocrine regulation are key indicators for evaluating the developmental status of male shrimp gonads. After sexual differentiation, the gonadal development stage of male shrimp can be divided into four periods, during which the morphology of the gonadal tissue and the number of sperm in the spermatophore of male shrimp change significantly. Among them, spermatogenesis is divided into three independent stages. The first is testis maturation, when immature sperm are produced. The second is vas deferens maturation, which completes the maturation of sperm through the formation of spines. Finally, spermatophore synthesis is carried out in the ampulla, and mature sperm are finally stored in the spermatophore. Studies have pointed out that sperm quality not only affects the success rate of fertilization, but also has an important impact on the developmental quality of offspring. Therefore, it is crucial to clarify the specific mechanism behind spermatogenesis. During the development of male shrimp gonads, it is currently believed that male shrimp are regulated by the endocrine axis of "eyestalk-androgenic gland (AG)-testis". AG is connected to the proximal part of the vas deferens and attached to the posterior vas deferens of male decapods, and is a key factor in regulating primary and secondary sexual characteristics. AG controls behavior, spermatogenesis and sexual differentiation by secreting insulin-like androgenic hormone (IAG), and its function is regulated by upstream factors such as glucagon (CHH), gonadal inhibitory hormone (GIH) and ecdysone inhibitor (MIH). Insulin-like receptors are its downstream limiting factors. These studies show that endocrine regulation is closely related to spermatogenesis. Although the relevant processes of sperm performance and endocrine regulation have been preliminarily described, systematic studies on how nutritional factors affect sperm through endocrine pathways are still insufficient. More importantly, the interaction mechanism between upstream factors and downstream receptors involved in the endocrine regulation mechanism is still unclear. These research gaps not only limit the comprehensive understanding of the reproductive physiology of male shrimp, but also hinder the further development of related breeding technologies. Therefore, it is necessary to further explore these endocrine regulatory mechanisms to more comprehensively understand and optimize the reproductive capacity of male shrimp. Summary of the invention

[0005] The invention provides an application of freeze-dried squid powder, feed and a shrimp breeding method, 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] The invention discloses application of squid freeze-dried powder in preparing feed for promoting male shrimp gonad development.

[0008] The invention also discloses a feed for promoting the development of male shrimp gonads. In the shrimp feed containing the fish meal, 40% to 60% of the fish meal is replaced by squid freeze-dried powder.

[0009] In a preferred embodiment of the present invention, per kilogram of the feed includes the following raw materials by weight: 180-220g of fish meal, 160-220g of freeze-dried squid powder, 200-220g of whey protein, 140-160g of corn starch, 8-12g of fish oil, 20-25g of soybean oil, 4-6g of cholesterol, 0.8-1.2g of 2,6-di-tert-butyl-4-methylphenol, 15-25g of soybean lecithin, 8-12g of vitamin premix, 15-25g of mineral premix, 12-17g of monocalcium phosphate, 8-12g of choline chloride, 15-25g of sodium carboxymethyl cellulose, and 110-120g of microcrystalline cellulose.

[0010] Furthermore, each kilogram of the vitamin premix includes the following raw materials in parts by weight:

[0011] Vitamin A acetate 0.45-0.5g; active L-ascorbic acid-2-polyphosphate 30-40g; folic acid 0.15-0.2g; biotin 2-3g; riboflavin 2-4g; DL-calcium pantothenate 3-7g; pyridoxine 0.8-1.2g; phenanthamine hydrochloride: 0.3-0.7g; vitamin B 12 0.1-0.3g; Vitamin K31-3g; Vitamin D30.6-1g; DL-α-tocopheryl acetate5-10g; Niacin4-6g; Defatted rice bran900-950g;

[0012] The concentration of vitamin A acetate is 500,000 IU / g, the concentration of L-ascorbic acid-2-polyphosphate is 35%, the concentration of vitamin D3 is 500,000 IU / g, and the concentration of DL-α-tocopherol acetate is 2.5 million IU / g.

[0013] Furthermore, each kilogram of the mineral premix includes the following raw materials in parts by weight:

[0014] Zinc sulfate monohydrate 22-25g; calcium iodate 0.1-0.2g; copper sulfate pentahydrate 0.5-0.7g; manganese sulfate monohydrate 1-2g; magnesium sulfate monohydrate 38-43g; cobalt chloride 0.005-0.015g; ferrous sulfate monohydrate 10-12g; sodium selenite 0.02-0.03g; calcium hydrogen phosphate dihydrate 160-170g; bran flour 700-800g.

[0015] The invention also discloses the use of the feed in promoting the development of male shrimp gonads. The feed is used to increase the total sperm count of male shrimp and reduce the abnormal sperm ratio.

[0016] Furthermore, the feed is used to increase the levels of IAG and MF in male shrimp serum and to increase the level of cell cycle dependent kinase CDK.

[0017] The present invention also discloses a method for culturing shrimps using the feed as described above, comprising the following steps:

[0018] Select male shrimps whose glandular development has reached stage III and feed them temporarily for 7 days with conventional feed;

[0019] After the temporary rearing, 100% of the water is changed every day, and six feedings are implemented, with each feeding amount being 5-6% of the shrimp body weight, for breeding.

[0020] The present invention also discloses differential metabolites for promoting gonadal development of male shrimp based on squid freeze-dried powder, including DHA, riboflavin, fructose-1,6-diphosphate, N-acetyl-α-neuraminic acid, 3-hydroxymethylglutaric acid, D-mannose, selegiline, all-trans retinoic acid, L-cystathionine and o-phosphoethanolamine.

[0021] The present invention also discloses the application of the differential metabolites mentioned above in detecting the gonadal development of male shrimps.

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

[0023] The present invention provides conclusive evidence for the significant effect of squid freeze-dried powder as a high-quality feed protein source in promoting the growth and gonadal development of male broodstock of Penaeus vannamei. It also preliminarily reveals its molecular mechanism for regulating the growth and development of male shrimp through specific metabolic pathways, providing important scientific basis and technical support for the efficient and sustainable development of aquaculture. 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 adding different biological bait freeze-dried powder to the feed on the growth performance of male Penaeus vannamei; (A) weight gain, (B) specific growth rate, (C) survival rate, (D) fatness. The values ​​in the bar graph are expressed as mean ± standard error (n = 4). Different letters in the same bar graph indicate significant differences between groups. (P < 0.05, one-way ANOVA and Duncan test);

[0026] Figure 2Effects of adding different biological bait freeze-dried powder to feed on the antioxidant status and nonspecific immunity of male Penaeus vannamei; (A) total antioxidant capacity, (B) glutathione peroxidase, (C) malondialdehyde, (D) phenol oxidase, (E) acid phosphatase, (F) alkaline phosphatase. The values ​​in the bar graph are expressed as mean ± standard error (n = 4). Different letters in the same bar graph indicate significant differences between groups. (P < 0.05, one-way analysis of variance and Duncan test);

[0027] Figure 3 Morphological analysis of the ampulla of male Penaeus vannamei fed with freeze-dried powder of different biological baits; (A, a) control group, (B, b) squid freeze-dried powder group, (C, c) lugworm freeze-dried powder group, (D, d) Antarctic krill freeze-dried powder group, (E) hepatopancreas index, (F) gonad index. The magnification used for observation was 400 times;

[0028] Figure 4 Histological analysis of the testes and vas deferens of male Penaeus vannamei fed with different biological bait freeze-dried powder feeds; (A, E) control group, (B, F) squid freeze-dried powder group, (C, G) lugworm freeze-dried powder group, (D, H) Antarctic krill freeze-dried powder group. Primary spermatocytes (sy1), secondary spermatocytes (sy2) and spermatids (spe). Structure of the middle vas deferens: primary spermatocyte layer (psl), sperm mass (sm), late spermatids (lsd);

[0029] Figure 5 Effects of adding freeze-dried powder of different biological baits to feed on sperm performance of male Penaeus vannamei; (A) Observation of sperm morphology at 100 times magnification during sperm counting. (B, C) Observation of sperm morphology at 400 times magnification during sperm counting. (D) Sperm of different morphologies. (E) Total sperm count, (F) Abnormal sperm ratio, (G) Sperm survival rate, (H) Spermatozoa weight. Sperm morphology: normal sperm (NS), dead sperm (NS) and sperm with malformed head (MHS), sperm with malformed tail: bent tail (ATS1), short tail (ATS2) and missing tail (ATS3). The values ​​in the bar graph are expressed as mean ± standard error (n = 12). Different letters annotated in the same bar graph indicate significant differences between groups. (P < 0.05, one-way ANOVA and Duncan test);

[0030] Figure 6Effects of different biological bait freeze-dried powder feeds on hormones and enzyme activities related to gonadal development in male Penaeus vannamei; (A) insulin-like androgen, (B) methyl farnesate, (C) hyperglycemic hormone, (D) gonadal inhibitory hormone, (E) molting inhibitory hormone, (F) cell cycle-dependent kinase. The values ​​in the bar graph are expressed as mean ± standard error (n = 8). Different letters in the same bar graph indicate significant differences between groups. (P < 0.05, one-way analysis of variance and Duncan test);

[0031] Figure 7 Effects of adding different biological bait freeze-dried powder to feed on the expression of genes related to gonadal development in male Penaeus vannamei; (A) Relative expression of insulin-like androgen, (B) Relative expression of insulin-like peptide, (C) Relative expression of insulin receptor, (D) Relative expression of guanylate cyclase, (E) Relative expression of cyclin-dependent kinase 2, (F) Relative expression of hyperglycemic hormone. The values ​​in the bar graph are expressed as mean ± standard error (n = 4). Different letters in the same bar graph indicate significant differences between groups (P < 0.05, one-way analysis of variance and Duncan's test);

[0032] Figure 8 Principal component analysis and number of differential metabolites in each group: (A) Chemical classification ratio of metabolites; (B) OPLS-DA score in positive ion mode; (C) OPLS-DA score in negative ion mode; (D) Differences in up-regulated and down-regulated metabolites in the experimental group compared with the control group;

[0033] Fig. 9 Screening analysis of common and unique differential metabolites; (A) Venn diagram analysis of differential metabolites in the three treatment groups. (B) Analysis of the expression levels of common differential metabolites in the three treatment groups and the expression levels of unique differential metabolites in the SFM group. (C) Overall expression of metabolites in each experimental group in all samples. (D) Analysis of differential ploidy of the expression of significant differential metabolites in the SFM group. (E) Analysis of differential ploidy of the expression of significant differential metabolites in the SFM group. (F) Analysis of differential ploidy of the expression of significant differential metabolites in the SFM group.

[0034] Fig.10 KEGG enrichment analysis of significantly differential metabolites in the SFM group; (A) KEGG pathway enrichment analysis of differential metabolites; (B) Network diagram of differential metabolites and enriched pathways; (C) Correlation heat map of differential metabolites. 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] The present invention mainly adopts low-temperature freeze-drying technology to make freeze-dried powder of three commonly used biological baits, replace 50% of fish meal in the control group, and make 4 new artificial compound feeds with equal nitrogen and equal fat. This technology aims to retain the nutrients and special active substances of the raw materials to the greatest extent. Through multi-level indicators such as gonadal external morphology, histology, antioxidant and immune status, endocrine regulation, and gonadal development-related genes, the effects of freeze-dried powder of different biological baits on the growth performance, endocrine regulation, and gonadal development of male broodstock are comprehensively evaluated, and the similarities and differences of freeze-dried powder of different biological baits in the cultivation effect are explored. In addition, through further metabolomics analysis, potential marker metabolites that affect the gonadal development of male broodstock are excavated, which provides a certain scientific basis for the cultivation of male broodstock and the research and development of artificial compound feeds for male broodstock.

[0037] The following is an explanation through specific embodiments.

[0038] Example 1 Preparation of experimental feed

[0039] According to the nutritional requirements of male Penaeus vannamei, the present invention prepares four experimental feeds with equal nitrogen (45% crude protein) and equal fat (8% crude fat). The control group uses fish meal and whey protein as the main protein sources, containing 400g / kg of fish meal. The experimental group uses equal amounts of squid freeze-dried powder, nemophore freeze-dried powder and Antarctic krill freeze-dried powder to replace 50% of the fish meal to prepare the other three feeds, which are named SFM, PFM and KFM respectively. The three freeze-dried powders are freeze-dried by vacuum freeze drying machine (SCIENTZ-18N, Ningbo Xinzhi Biotechnology Co., Ltd., China) fresh squid (Uroteuthisduvaucelii), nemophore (Perinereis aibuhitensis) and Antarctic krill (Euphausia superba), and then crushed by a pulverizer and passed through a 60-mesh screen to make powder. Other granular raw materials are also crushed by a pulverizer and passed through a 60-mesh screen to make powder. All processed feed ingredients were weighed according to the formula ratio and placed in a container, and then mixed thoroughly using an electric mixer. Then oil and water were added and mixed evenly using an electric mixer again. After that, the feed mixture was made into feed pellets with a diameter of 2.5 mm by a double-screw plodder (CD4-1TS, Guangdong Huagong Opto-Mechanical Technology Co., Ltd., China) and air-dried at room temperature. Before use, the pellet feed was sealed in a plastic bag and stored at -20 °C.

[0040] The main raw materials and approximate composition of the feed were determined by standard methods. The experimental feed was dried to constant weight at 105°C, then weighed and the moisture content was calculated; crude protein content was determined by Dumas nitrogen analyzer (RAPID N EXCEED, ELEMENTAR, Germany); Soxhlet extraction was used for crude fat determination; ash content was determined after burning at 550°C for 10 h in a muffle furnace. Table 1 shows the detailed information of the feed formula and its composition.

[0041] Table 1 Composition (g / kg dry basis) and approximate composition of the four experimental feeds

[0042]

[0043] 1. Vitamin premix (g / kg premix): Vitamin A acetate (500,000 IU / g): 0.480; 35% active L-ascorbic acid-2-polyphosphate: 35.710; folic acid: 0.180; biotin: 2.500; riboflavin: 3.000; DL-calcium pantothenate: 5.000; pyridoxine: 1.000; phenanthroline hydrochloride: 0.500; vitamin B 12 :0.200; Vitamin K3:2.000; Vitamin D3 (500,000 IU / g):0.800; DL-α-tocopheryl acetate (250 IU / g):8.000; Niacin:5.000; Defatted rice bran:935.630.

[0044] 2. Mineral premix ( / kg premix): zinc sulfate monohydrate: 20.585; calcium iodate: 0.117; copper sulfate pentahydrate: 0.625; manganese sulfate monohydrate: 1.625; magnesium sulfate monohydrate: 39.860; cobalt chloride: 0.010; ferrous sulfate monohydrate: 11.179; sodium selenite: 0.025; calcium hydrogen phosphate dihydrate: 166.442; bran flour: 759.532.

[0045] Example 2 Experimental shrimp farming

[0046] The present invention uses 328 male Penaeus vannamei broodstock from a local private enterprise in Hainan. Male shrimps with gonad development to stage III are selected as experimental subjects. The changes in gonad morphology and the number of sperm in spermatophores during the development of male shrimps in this period to mature stage IV are most significant. The initial average body weight of male shrimps is 32.39±0.18g, and the average body length is 13.74±0.48cm. Eight of them are randomly selected to measure the initial hepatopancreas index (HSI, 4.123±0.189%) and gonad index (GSI, 0.535±0.031%). Subsequently, the shrimps are randomly divided into 16 barrels (bottom diameter×height=1m×0.9m), and each experimental group is set up with four replicates, with a density of 20 per barrel. Before the experiment begins, they are temporarily raised in the experimental barrel for 7 days, during which they are fed with control group feed to adapt to the experimental conditions. During the experiment, 100% of the water was changed every day, and six feedings were implemented (7:30AM, 11:30AM, 3:00PM, 6:30PM, 9:30PM, 11:30PM), and the feeding amount each time was 5.5% of the shrimp body weight. The nutritional experiment period was 28 days.

[0047] Test example

[0048] After the experimental shrimp culture of Example 2 was completed, 15 shrimps were randomly selected from each bucket of each experimental group. First, three shrimps were randomly selected to observe the characteristics of the ampulla under a stereomicroscope (STZ-02, Shanghai Optical Instrument Factory No. 1, China), followed by dissection, and the characteristics of the testes and vas deferens after dissection were photographed. The remaining 12 shrimps were anesthetized in ice water, and their body length and weight were recorded. Hemolymph was extracted from the heart cavity and abdomen (first pair of swimming feet) using a 1mL sterile syringe and stored at 4°C for 24h. Subsequently, the shrimp bodies were dissected, the eyestalks, hepatopancreas, and gonads were collected, and the gonads were separated into testes and vas deferens and placed in cryopreservation tubes, frozen in liquid nitrogen, and then stored at -80°C for subsequent analysis. Some of the testes and vas deferens were fixed in 4% paraformaldehyde, and histological observation was performed by hematoxylin-eosin (H&E) staining in paraffin tissue sections. The calculation formulas for weight gain rate (WG), specific growth rate (SGR), survival rate (SR), fatness (FC), hepatopancreas index (HSI), and gonadal index (GSI) are as follows:

[0049] (1) Weight gain rate (%) = (final weight - initial weight) / initial weight × 100

[0050] (2) Specific growth rate (%, day) -1 ) = [In (final body weight) - In (initial body weight)] / number of days of culture × 100

[0051] (3) Survival rate (%) = (final number / initial number) × 100

[0052] (4) Fullness (g / cm3 ) = final body weight / (body length) 3 ×100

[0053] (5) Hepatosomatic index (%) = (hepatopancreas wet weight / wet body weight) × 100

[0054] (6) Gonad index (%) = (gonad wet weight / wet body weight) × 100

[0055] Data analysis: Spearman correlation analysis was performed using SPSS software (version 27.0, SPSS Inc., USA). The heat map shows the correlation and significant difference between the marker metabolites and the growth, antioxidant capacity, gonadal development and sperm performance related indicators of male shrimp. "*" indicates a significant correlation (P < 0.05), and "**" indicates an extremely significant correlation (P < 0.01). All data in the study were subjected to one-way analysis of variance (ANOVA) in SPSS (ver. 27.0; SPSS Inc., USA), and then Duncan multiple calculations were performed to determine the significance between all experimental groups. The experimental results are expressed as mean ± standard error, and P < 0.05 indicates a significant difference.

[0056] 1. Investigate the growth performance of shrimp in different experimental groups

[0057] The growth of male broodstock of Penaeus vannamei treated with different freeze-dried biological baits is shown in the following figure. Figure 1 As shown. Figure 1 It can be seen that compared with the control group, the experimental groups fed with three different biological bait freeze-dried powders significantly increased the weight gain rate and specific growth rate ( Figure 1 , AB, P<0.05). Among them, the weight gain rate of the SM group was significantly higher than that of the other three groups ( Figure 1 , AB, P<0.05). Although there was no significant difference in weight gain rate between the PFM and KFM groups, the specific growth rate of the KFM group was significantly higher than that of the PFM group. No significant difference was found in the survival rate among the experimental groups ( Figure 1 , C, P>0.05). It is worth noting that the fatness of the SFM group was the lowest among all experimental groups ( Figure 1 , D, P<0.05), but there was no significant difference compared with the KFM group ( Figure 1 , D, P>0.05).

[0058] 2. Investigate the antioxidant capacity and immune capacity of shrimp in different experimental groups

[0059] Eight hepatopancreas, eyestalk, and testis samples were randomly selected from each experimental group, weighed, and transferred to a 1.5 ml centrifuge tube. Nine volumes of 0.86% saline (1:10, w / v) were added, and the samples were homogenized at 60 Hz for 1 min using a tissue grinder (Tissuelyser-24, Jingxin Technology, China). The tissues were then centrifuged at 3000 r / min for 10 min at 4°C using a centrifuge (3-18K; Sigma, Germany). After centrifugation, the supernatant was collected for subsequent analysis. At the same time, eight hemolymph samples that had been placed at 4°C for 24 h were randomly selected from each experimental group and crushed. The samples were then centrifuged at 3000 r / min for 15 min using a centrifuge, and the supernatant was collected for subsequent analysis.

[0060] The pretreated hepatopancreas supernatant was used to detect the total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels. The hemolymph supernatant was used to determine the levels of phenoloxidase (PO), acid phosphatase (ACP), and alkaline phosphatase (AKP). All tests were performed using Nanjing Jiancheng kits, and all tests were performed in accordance with the instructions provided by the manufacturer.

[0061] Feeding freeze-dried powder feeds supplemented with different biological baits had significant effects on the antioxidant capacity and immune parameters of male Penaeus vannamei. Figure 2 As shown. Figure 2 It can be seen that compared with the control group, the total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) activity of the hepatopancreas in the SFM and KFM groups were significantly increased ( Figure 2 , AB, P<0.05). Among the three treatment groups, the GSH-Px level in the KFM group was significantly higher than that in the PFM group ( Figure 2 , B, P<0.05), while the total antioxidant capacity and GSH-Px activity of the SFM group were not significantly different from those of the KFM group. Compared with the control group, the malondialdehyde (MDA) level of each treatment group was significantly reduced ( Figure 2 , C, P<0.05), among which the MDA levels of the SFM and KFM groups were significantly lower than those of the PFM group, but there was no significant difference between the SFM and KFM groups.

[0062] The activities of phenol oxidase, acid phosphatase, and alkaline phosphatase were the highest in the SFM group. The phenol oxidase level in the SFM group was significantly higher than that in the control group and the PFM group ( Figure 2 , D, P<0.05). In terms of acid phosphatase activity, the SFM group was significantly higher than the other three experimental groups ( Figure 2 , E, P<0.05), but there was no significant difference between the PFM and KFM groups. The alkaline phosphatase level in the SFM group was significantly higher than that in the PFM group ( Figure 2 , F, P<0.05), there was no significant difference in the alkaline phosphatase level between the SFM group and the control group and the KFM group ( Figure 2 , F, P>0.05).

[0063] 3. Investigate the gonadal development of shrimp in different experimental groups

[0064] For gonadal histological analysis, the testicular lobe and the middle vas deferens fixed in 4% paraformaldehyde for 24 h were first dehydrated with gradient alcohol and washed. Then, ethanol, toluene and xylene were balanced, then embedded in paraffin and cut into 4 μm sections using a rotary microtome (Leica RM2016, Germany). After sectioning, hematoxylin and eosin (H&E) were used for staining, and the histological characteristics of the obtained sections were observed under an optical microscope (ECLIPSE 200, Nikon, Japan).

[0065] In terms of gonadal index, the results were as follows Figure 3 As shown. Figure 3 It can be seen that the SFM group performed particularly well, reaching the highest level, significantly higher than the other experimental groups ( Figure 3 F, P<0.05). However, the hepatopancreatic index of the SFM group was the lowest among the three experimental groups and was significantly lower than that of the other two experimental groups ( Figure 3 E, P<0.05). In order to more intuitively evaluate the gonadal development of male shrimp, the morphological observation of the ampulla was carried out, focusing on the fullness and color of the ampulla. Compared with the control group, the ampulla of each experimental group was fuller, among which the fullness of the ampulla of the SFM group was the most significant, and it was milky white. The ampulla of the PFM group had a slight yellow pigmentation, while the yellow pigmentation of the ampulla of the KFM group was the most obvious. There was no significant difference in the morphology of the vas deferens among the four groups ( Figure 3 ). At the same time, the histological analysis of testis and vas deferens was Figure 4 The results showed that the testicular cells of each experimental group mainly included primary spermatocytes, secondary spermatocytes and spermatids, among which the proportion of spermatids and primary spermatocytes in the testis of the SFM group was significantly higher than that of the other groups. The main structures of the vas deferens in all experimental groups were sperm masses, primary spermatocyst layers and spermatocyst layers, and the distribution area of ​​the spermatocyst layer was significantly larger than that of the other three groups.

[0066] 4. Investigate the sperm performance of shrimps in different experimental groups

[0067] Sperm quality was evaluated by spermatophore weight, total sperm count, survival rate, and abnormal proportion. Three male broodstock were randomly selected from each bucket of each experimental group, with a total of 12 in each group. Two spermatophores of each shrimp were squeezed out of the male body by gently pressing the ampulla of the fifth leg, and the weight was recorded. After weighing, the two spermatophores of each shrimp were placed in a grinder, 2 mL of calcium-free artificial seawater was added, and the sperm was fully ground using a glass rod to ensure that the sperm was completely released into the solution and evenly mixed. 0.9 mL of sperm solution was taken, 0.1 mL of 1% trypan blue solution (1 g trypan blue was dissolved in 100 mL of calcium-free artificial seawater and the pH was adjusted to 7.4) was added, and dyed for 15 minutes. Finally, 12 1 mL sperm suspension samples were prepared for each group. The total number of sperm and the number of different types of sperm were counted using a hemacytometer and an optical microscope, and each sample was observed four times. Stained sperm were considered dead sperm, while unstained sperm were considered live sperm. For abnormal sperm counts, there are mainly different types: head deformity, spinous process bending, short or missing. Sperm survival rate and sperm abnormality ratio are calculated as follows:

[0068] (1) Sperm survival rate (%) = (number of live sperm / total number of sperm) × 100

[0069] (2) Sperm abnormality ratio (%) = (abnormal sperm count / total sperm count) × 100

[0070] The results are as follows Figure 5 As shown in Figure 1, when observed under a microscope at 100 times magnification, only the number of sperm can be counted, and their specific morphology cannot be identified ( Figure 5 , A). After increasing the magnification to 1000 times, it was found during the statistical process that sperm can be divided into six types: normal sperm, dead sperm, head deformity, tail bending, short tail and tail absence. Normal sperm are characterized by a plump head and a long straight tail spine, while dead sperm appear blue ( Figure 5 , BD). In terms of sperm count, the SFM group was significantly higher than the other experimental groups ( Figure 5 , E, P<0.05). In terms of sperm survival rate, among the three treatment groups, the sperm survival rates of the SFM and KFM groups were significantly higher than those of the PFM group ( Figure 5 , F, P<0.05). In addition, the SFM group had the lowest sperm abnormality rate, which was significantly lower than all other experimental groups, while the KFM group had the highest sperm abnormality rate, which was significantly higher than other experimental groups ( Figure 5 , G, P<0.05). The spermatophore weight of the SFM group was the smallest, and there was no significant difference in spermatophore weight among all experimental groups ( Figure 5 , H, P>0.05).

[0071] 4. Investigate the content of endocrine hormones and enzyme activities related to gonadal development in different experimental groups

[0072] The concentrations of hyperglycemic hormone (CHH), gonadal inhibitory hormone (GIH), and molting inhibitory hormone (MIH) in the supernatant of the eyestalk grinding were determined using ELISA kits (Shanghai Fantai Biological Co., Ltd., China). The hemolymph and testicular supernatants were used to determine insulin-like androgenic hormone (IAG), methyl farnesate (MF), and cell cycle-dependent kinase (CDK), respectively. All reagents and operations strictly followed the manufacturer's instructions of Shanghai Fantai Biological Co., Ltd. The standard curve was prepared using standards of known concentrations, and the absorbance of the samples was compared with that of the standards using a linear regression equation. The original concentration of the hormone was calculated by the formula and adjusted according to the dilution factor to obtain the actual concentration. The results are shown in Figure 6 shown.

[0073] Figure 6 The effects of adding different biological bait freeze-dried powder to feed on the endocrine hormone content and related enzyme activities of male Penaeus vannamei were shown. Compared with the control group, the levels of IAG and MF in the serum of the three treatment groups were significantly increased, among which the levels of these two hormones in the SFM group were the highest, significantly higher than those in the KFM group ( Figure 6 , AB, P<0.05). It is worth noting that the CHH level in the SFM group was significantly lower than that in the control group and other treatment groups ( Figure 6 , C, P<0.05). In addition, compared with the control group, the levels of GIH and MIH in the three treatment groups were significantly decreased ( Figure 6 , DE, P<0.05). Although the levels of GIH and MIH were the lowest in the SFM group, there were no significant differences among the three treatment groups. In addition, cell cycle-dependent kinases (CDKs) play a vital role in cell cycle regulation and are essential for the meiosis of spermatocytes and oocytes. This study detected the levels of CDKs in each group. The results showed that among the three treatment groups, the CDK level in the SFM group was the highest, significantly higher than that in the PFM group ( Figure 6 , F, P<0.05), but there was no significant difference with the KFM group.

[0074] 5. Investigate the gene expression related to gonadal development in male shrimps of different experimental groups

[0075] (1) RNA extraction and real-time fluorescence quantitative PCR

[0076] Real-time fluorescence quantitative PCR was used to quantitatively analyze the expression levels of CHH, GC, CDK2, IAG, ILP, and IR. Eight samples including eyestalks, testes, and distal vas deferens (including terminal ampulla) were randomly selected from each experimental group. The eyestalks were used to determine the expression level of CHH, the testes were used to detect the expression levels of GC and CDK, and the distal vas deferens (including terminal ampulla) was used to determine the expression levels of IAG, ILP, and IR. After total RNA was extracted using TRIzol reagent (GLPBIO, USA), its concentration and quality were evaluated using NanoDrop2000 spectrophotometer and 1.5% agarose gel electrophoresis. Subsequently, PrimeScriptTM RTReagent kits (RR047A, Takara, Japan) were used to convert total RNA into cDNA, and fluorescence quantitative analysis was performed on the The results were performed on a 96-well system (Roche, Switzerland) using ChamQ Universal SYBR qPCR Master Mix (Q711-03, Vazyme, China). The primers used for qPCR are shown in Table 2. -ΔΔCt Perform analytical calculations.

[0077] Table 2 Primer sequences used for quantitative real-time PCR (qPCR)

[0078]

[0079] IAG, insulin-like androgenic hormone; ILP, insulin-like peptide; IR, insulin receptor; GC, guanylate cyclase; CDK2, cyclin-dependent kinase 2; CHH, hyperglycemic hormone.

[0080] (2) After feeding the feed supplemented with freeze-dried powder of different biological baits, the expression of genes related to gonadal development in male shrimps was Figure 7 The mRNA expression of IAG and ILP in the vas deferens (including the terminal ampulla) was the highest in the SFM group, significantly higher than that in the other three groups (P<0.05), while there was no significant difference between the PFM group and the KFM group ( Figure 7 , AB, P<0.05). In addition, compared with the control group, the mRNA expression levels of IR in all treatment groups were significantly increased, among which the expression level in the SFM group was significantly higher than that in the other two treatment groups ( Figure 7 , C, P<0.05). In addition, compared with the control group, the mRNA expression level of CDK-2 in the testis of all treatment groups was significantly increased ( Figure 7, E, P<0.05), among which the expression level of SFM group was the highest, significantly higher than that of other treatment groups, followed by KFM group, and there was no significant difference between PFM group and KFM group. On the contrary, the mRNA level of GC in SFM group was the lowest, significantly lower than that in control group and PFM group ( Figure 7 , D, P<0.05), while there was no significant difference between the PFM group and the KFM group. Similarly, the mRNA expression level of CHH in the eyestalk was the lowest in the SFM group, which was significantly lower than that in the control group and the PFM group ( Figure 7 , F, P<0.05), while there was no significant difference between the PFM group and the KFM group.

[0081] 6. Serum metabolomics and correlation analysis

[0082] In order to deeply analyze the nutritional metabolic characteristics of each experimental group, 4 serum samples were randomly selected from each group, and the differences in serum metabolites were analyzed on the Shanghai Paisono biological platform. First, the metabolites were extracted. 25 mg of the sample was weighed and placed in an EP tube under low temperature conditions. Homogenizing beads and 500 μL of extract containing isotope-labeled internal standards were added, vortexed for 30 seconds, homogenized in a homogenizer at 35 Hz for 4 minutes, and then transferred to an ice water bath for ultrasonication for 5 minutes. This process was repeated 3 times and allowed to stand at -40 ° C for 1 hour; centrifuged at 4 ° C and 12000 rpm for 15 minutes. An equal amount of supernatant was taken from all samples and mixed into the quality control sample for testing to ensure the stability of the experiment. For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph. The target compounds were chromatographically separated by a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) liquid chromatography column. Phase A of the liquid chromatography is an aqueous phase containing 25mmol / L ammonium acetate and 25mmol / L ammonia water, and phase B is acetonitrile. The XCMS software package of R was used for peak detection, peak filtering, and peak alignment to obtain a quantitative list of substances. Subsequent metabolite identification and analysis were then performed. Multivariate statistical analysis was performed using orthogonal partial least squares discriminant analysis (OPLS-DA), and thresholds were set for differential metabolite screening (VIP>1, P value<0.05), and differential metabolites of the three comparison groups (SFM vs Ctrl, PFM vs Ctrl, KFM vs Ctrl) were identified respectively. The marker metabolites were further identified by Venn diagram analysis. In addition, based on the fact that the SFM group performed best among all groups, the important pathways of its differential metabolites were also analyzed. The relationship between metabolites and enriched pathways was analyzed by a network diagram of differential metabolites and enriched pathways. The results are as follows. Figure 8-10 shown.

[0083] A total of 287 metabolites were identified in the present invention, of which 189 were identified in positive ion mode (POS) and 98 were identified in negative ion mode (NEG). According to chemical classification, the metabolites are mainly divided into fatty acyl, carboxylic acid and its derivatives, organic oxygen compounds, benzene and substituted derivatives, and steroids and steroid derivatives ( Figure 8 , A). OPLS-DA analysis of serum metabolic profiles showed that samples of all groups were within the 95% confidence ellipse ( Figure 8 , BC). Compared with the control group, a total of 48 differential metabolites were screened in the three experimental groups. Among them, there were 5 up-regulated metabolites and 9 down-regulated metabolites in the SFM group. The up-regulated metabolites included: docosahexaenoic acid (DHA), N-acetyl-α-neuraminic acid, riboflavin, Se-methylselenocysteine, fructose-1,6-diphosphate and 3-hydroxymethylglutaric acid. The down-regulated metabolites mainly included all-trans retinoic acid, L-cystathionine, o-phosphoethanolamine, γ-tocotrienol, α-tocotrienol and guanosine-5'-triphosphate. There were 7 up-regulated metabolites and 5 down-regulated metabolites in the PFM group. There were 7 up-regulated metabolites and 5 down-regulated metabolites in the PFM group. The up-regulated metabolites mainly included DHA, deoxycholic acid, riboflavin, lanosterol, spermidine, 4-hydroxycinnamic acid and fructose-1,6-bisphosphate; the down-regulated metabolites mainly included D-phenylalanine, 6-hydroxyhexanoic acid, 2-ketobutyric acid and vitamin D3. There were 9 up-regulated metabolites and 13 down-regulated metabolites in the KFM group. The up-regulated metabolites were mainly DHA, deoxycholic acid, spermidine, riboflavin, Se-methylselenocysteine, lanosterol, 4-hydroxycinnamic acid and fructose-1,6-bisphosphate; the down-regulated metabolites were mainly oleoylethanolamide, γ-tocotrienol, protoporphyrin IX, aspartame, α-tocotrienol, guanosine-5'-triphosphate and stigmasterol ( Figure 8 , D; Fig. 9 , DF). The detailed information of the differential metabolites of all comparison groups is shown in Table 3.

[0084] Table 3 Different metabolites in three different comparison methods

[0085]

[0086]

[0087]

[0088]

[0089] Through the Venn diagram analysis of the differential metabolites of the three comparison groups, it was found that there were three common differential metabolites in the three groups, namely: DHA, riboflavin and fructose-1,6-bisphosphate. There were seven unique differential metabolites in the SFM group, namely: N-acetyl-α-neuraminic acid, 3-hydroxymethylglutaric acid, D-mannose, selegiline, all-trans retinoic acid, L-cystathionine and o-phosphoethanolamine ( Fig. 9 , A). Further analysis of the expression levels of these metabolites in each experimental group showed that compared with the control group, the levels of DHA, riboflavin and fructose-1,6-bisphosphate in the serum of the three treatment groups were significantly increased, but there was no significant difference among the three experimental groups ( Fig. 9 , B).

[0090] Given that the performance of the SFM group was the most prominent, the pathways of the differential metabolites in the SFM group were further analyzed. The results of KEGG enrichment analysis are shown in Fig.10 As shown in the figure, compared with the control group, a total of 29 metabolic pathways were identified in the SFM group, among which the most significantly enriched ones were riboflavin metabolism, ABC transporters, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, lysosomes, autophagy-animals, endocytosis, ubiquinone and other terpene quinone biosynthesis, cofactor biosynthesis, and sulfur transport system. The relationship between these highly enriched pathways was analyzed by metabolic network diagram ( Fig.10 , A), found that ABC transporters, riboflavin metabolism, lysosomes, autophagy-animals, endocytosis, biosynthesis of cofactors, and sulfur transport systems were interconnected through L-cystathionine, riboflavin, protoporphyrin IX, and D-mannose in the SFM group. ( Fig.10 , B)

[0091] Correlation analysis of the 10 differential metabolisms screened above showed that all-trans retinoic acid was significantly positively correlated with MIH (P<0.05), and significantly negatively correlated with Lv-ILP, Lv-IAG and Lv-IR (P<0.05). D-mannose was significantly positively correlated with HSI, AS and CHH (P<0.05), and significantly negatively correlated with CDK, GSI, TSC and Lv-IAG (P<0.05). In addition, D-mannose was extremely significantly negatively correlated with MF, WG, Lv-CDK2 and Lv-IR (P<0.01). L-cystathionine was extremely significantly positively correlated with MIH, GIH and Lv-GC (P<0.05), significantly positively correlated with FC, and extremely significantly negatively correlated with MF, WG, Lv-IR and Lv-IAG (P<0.01). N-acetyl-α-neuraminic acid was significantly positively correlated with T-AOC, PO and ACP (P<0.05), and extremely significantly positively correlated with GSI, CDK, GSW, WG, MF and Lv-CDK2 (P<0.01). In addition, N-acetyl-α-neuraminic acid was significantly negatively correlated with MDA and FC, and extremely significantly negatively correlated with CHH and Lv-CHH (P<0.01). Riboflavin was extremely significantly positively correlated with HSI. DHA and fructose-1,6-bisphosphate were significantly positively correlated with IAG, respectively (P<0.05). ( Fig.10 , C)

[0092] In summary, the present invention found that compared with the freeze-dried powder groups of sandworms and Antarctic krill, the male shrimps in the freeze-dried powder group of squid showed the best growth performance and maturity. Squid freeze-dried powder can significantly increase the total sperm count and reduce the proportion of abnormal sperm, and has the best sperm performance. This is attributed to the fact that the squid freeze-dried powder can regulate the production and secretion of hormones in the upstream eyestalks of male shrimps, promote the production of IAG, and increase the expression of downstream IR, thereby playing a role in the male body and promoting sperm formation and gonadal development. In addition, metabolomics was used to clarify the metabolic characteristics of the optimal biological bait freeze-dried powder on the gonadal development of male shrimps, and multiple marker metabolites related to the gonadal development of male shrimps, such as docosahexaenoic acid (DHA), riboflavin, fructose-1,6-diphosphate, N-acetyl-a-neuraminic acid, L-cystathionine and o-phosphoethanolamine, were screened, laying a certain foundation for the future research and development of male broodstock maturation-promoting feeds.

[0093] 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 squid freeze-dried powder in the preparation of feed for promoting gonadal development of male shrimp.

2. A feed for promoting gonadal development of male shrimp, characterized in that: In shrimp feed containing fish meal, squid freeze-dried powder is used to replace 40% to 60% of the fish meal.

3. The feed for promoting gonadal development of male shrimp according to claim 2, characterized in that: Each kilogram of the feed includes the following raw materials by weight: 180-220g of fish meal, 160-220g of squid freeze-dried powder, 200-220g of whey protein, 140-160g of corn starch, 8-12g of fish oil, 20-25g of soybean oil, 4-6g of cholesterol, 0.8-1.2g of 2,6-di-tert-butyl-4-methylphenol, 15-25g of soybean lecithin, 8-12g of vitamin premix, 15-25g of mineral premix, 12-17g of monocalcium phosphate, 8-12g of choline chloride, 15-25g of sodium carboxymethyl cellulose, and 110-120g of microcrystalline cellulose.

4. The feed for promoting gonadal development of male shrimp according to claim 3, characterized in that: Each kilogram of the vitamin premix includes the following raw materials in parts by weight: Vitamin A acetate 0.45-0.5g; active L-ascorbic acid-2-polyphosphate 30-40g; folic acid 0.15-0.2g; biotin 2-3g; riboflavin 2-4g; DL-calcium pantothenate 3-7g; pyridoxine 0.8-1.2g; phenanthamine hydrochloride: 0.3-0.7g; vitamin B 12 0.1-0.3g; Vitamin K31-3g; Vitamin D30.6-1g; DL-α-tocopheryl acetate5-10g; Niacin4-6g; Defatted rice bran900-950g; The concentration of vitamin A acetate is 500,000 IU / g, the concentration of L-ascorbic acid-2-polyphosphate is 35%, the concentration of vitamin D3 is 500,000 IU / g, and the concentration of DL-α-tocopherol acetate is 2.5 million IU / g.

5. The feed for promoting gonadal development of male shrimp according to claim 3, characterized in that: Each kilogram of the mineral premix includes the following raw materials in parts by weight: Zinc sulfate monohydrate 22-25g; calcium iodate 0.1-0.2g; copper sulfate pentahydrate 0.5-0.7g; manganese sulfate monohydrate 1-2g; magnesium sulfate monohydrate 38-43g; cobalt chloride 0.005-0.015g; ferrous sulfate monohydrate 10-12g; sodium selenite 0.02-0.03g; calcium hydrogen phosphate dihydrate 160-170g; bran flour 700-800g.

6. Use of the feed according to any one of claims 2 to 5 in promoting gonadal development in male shrimps, characterized in that: The feed is used for increasing the total sperm count of male shrimps and reducing the abnormal sperm ratio.

7. The use according to claim 6, characterized in that: The feed is used for increasing the levels of IAG and MF in male shrimp serum and increasing the level of cell cycle dependent kinase CDK.

8. A method for culturing shrimp using the feed according to any one of claims 2 to 5, characterized in that: The steps include: Select male shrimps whose glandular development has reached stage III and feed them temporarily for 7 days with conventional feed; After the temporary rearing, 100% of the water is changed every day, and six feedings are implemented, with each feeding amount being 5-6% of the shrimp body weight, for breeding.

9. Differential metabolites based on squid freeze-dried powder to promote male shrimp gonadal development, characterized in that: Includes DHA, riboflavin, fructose-1,6-bisphosphate, N-acetyl-α-neuraminic acid, 3-hydroxymethylglutaric acid, D-mannose, selegiline, all-trans retinoic acid, L-cystathionine and o-phosphoethanolamine.

10. Use of the differential metabolites according to claim 9 in detecting gonadal development of male shrimp.

Images