Application of caffeic acid phenethyl ester high-fat feed in improvement of overwintering health condition of grass carp and reduction of fat loss rate

Abstract

The invention discloses an application of a caffeic acid phenethyl ester high-fat feed in improving the overwintering health condition of grass carp and reducing the fat loss rate, and belongs to the technical field of aquaculture. According to the method, the caffeic acid phenethyl ester is added into the high-fat feed, and the grass carp is fed within 40 days before overwintering. Experiments prove that according to the strategy, fat cells of the grass carps can be proliferated before overwintering, so that the energy utilization mode of the grass carps in the overwintering period is changed, the health condition of the grass carps in the overwintering period is improved, and finally the weight loss of the grass carps is reduced. New opinions and theoretical bases are provided for improving the health condition of the cultured fishes in the overwintering period and reducing fat loss of the cultured fishes in the overwintering period.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular belongs to application of a high-fat feed of caffeic acid phenethyl ester in improving the health condition of grass carp during winter and reducing the fat loss rate. Background Art

[0002] Grass carp (Ctenopharyngodon idellus) has the characteristics of fast growth, easy breeding difficulty, low breeding cost and delicious meat. It is one of the important bulk freshwater fish and also the farmed fish with the largest output in freshwater aquaculture. During the grass carp breeding process, it usually goes through at least one wintering period before it can be put on the market.

[0003] In aquaculture, when the water temperature is low in winter, the metabolism of fish slows down and they "stop" eating, and the farmers also stop feeding; however, the consequence is that after the winter, the weight of farmed fish will decrease (commonly known as fat loss), causing huge economic losses. In order to reduce the economic losses caused by fat loss during the winter, high-fat feed is usually used before the winter in production practice to reduce the fat loss of farmed fish. However, while this method reduces the fat loss of farmed fish during the winter, it also has a serious negative impact on the health of the fish, making it difficult to recover after the winter, and causing a large number of deaths.

[0004] Adipose tissue is the most important lipid storage site in animals and is specifically used to store excess energy. Studies have shown that the way in which adipose tissue expands is a key factor in determining whether obesity leads to metabolic disorders. When adipose tissue expansion occurs mainly through adipocyte hypertrophy (increase in adipocyte size), it will lead to adipocyte dysfunction, such as reduced oxygen supply and increased levels of hypoxia-inducible factor, which in turn promotes the production of proinflammatory mediators, which are usually associated with insulin resistance, hepatic steatosis, and metabolic dysfunction. In addition, hypertrophic adipocytes may not be able to mobilize and release lipids effectively, resulting in limited energy mobilization. On the contrary, in the case of excess energy, increasing the number of adipocytes through hyperplasia (lipogenesis) can promote the healthy expansion of adipose tissue and systemic metabolic health. Newly generated adipocytes usually have better metabolic activity, can store and mobilize energy more effectively, and are easier to regulate energy balance. In addition, studies have found that high-fat diets inhibit adipocyte hyperplasia, thereby inducing pathological expansion of excessive adipocyte hypertrophy in both mammals and fish. Therefore, feeding grass carp with high-fat diets before wintering can further activate adipocyte proliferation, which is expected to be an effective strategy to improve the health of grass carp during wintering and reduce fat loss. However, there are currently few substances that can promote adipocyte proliferation and are safe and reliable, and the added concentration and feeding strategy are also unknown. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a high-fat feed of caffeic acid phenethyl ester for use in improving the health status of grass carp during winter and reducing the fat loss rate. By feeding the grass carp with a high-fat feed containing 400 mg / kg of caffeic acid phenethyl ester for 40 days before wintering, the fat cells of the grass carp can expand in a hyperplastic manner, so that energy can be stored and mobilized more effectively, thereby improving the health status of the grass carp during wintering and reducing fat loss. This method can effectively improve the health status of the grass carp during wintering and reduce the weight loss during wintering.

[0006] To achieve the above object, the present invention provides the following technical solutions: The invention provides application of caffeic acid phenethyl ester in fish feed.

[0007] Furthermore, the fish is grass carp.

[0008] The invention provides a high-fat feed, which contains caffeic acid phenethyl ester.

[0009] Furthermore, the added amount of caffeic acid phenethyl ester is 400 mg / kg to 1200 mg / kg.

[0010] The invention provides a method for improving the health status of grass carp during winter and reducing the fat loss rate, wherein the grass carp is fed with the high-fat feed 40 days before wintering.

[0011] Furthermore, during the feeding period, the animals were fed artificially to satiety, three times a day at 9:00, 13:00, and 17:00, each feeding lasting 1 hour.

[0012] The invention provides a high-fat feed for grass carp, which contains caffeic acid phenethyl ester.

[0013] Furthermore, the added amount of caffeic acid phenethyl ester is 400 mg / kg to 1200 mg / kg.

[0014] The invention provides application of the above-mentioned high-fat feed for grass carp in improving the health condition of grass carp during winter and reducing the fat loss rate.

[0015] Furthermore, the grass carp is fed with the high-fat feed 40 days before the grass carp hibernates. During the feeding period, the grass carp is fed artificially to satiety, three times a day at 9:00, 13:00, and 17:00, each time for 1 hour.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention introduces caffeic acid phenethyl ester, a natural phenolic compound, into high-fat feed for grass carp, achieving significant improvement in the health of grass carp during wintering. Compared with traditional feed, this feed is more targeted and effective. Caffeic acid phenethyl ester, as a natural ingredient extracted from propolis, not only has excellent antioxidant properties, but also can effectively promote adipocyte proliferation and change the expansion pattern of adipose tissue under high-fat conditions. This innovative application not only enriches the research field of grass carp feed, but also provides new ideas and methods for improving the health of grass carp during wintering.

[0017] By feeding grass carp with the high-fat feed provided by the present invention, the energy storage, mobilization and utilization efficiency of adipose tissue during the winter can be significantly improved, thereby effectively alleviating health problems such as liver lesions during the winter. This effect is due to the addition of caffeic acid phenethyl ester, which promotes the proliferation of grass carp fat cells, allowing adipose tissue to better cope with the energy demand during the winter. At the same time, the feed can also significantly reduce the weight loss and fat loss rate of grass carp during the winter, maintain the body shape and weight of grass carp, and bring higher economic benefits to breeders.

[0018] The application method of the present invention is simple and easy to implement, and only needs to feed the grass carp with a high-fat feed containing caffeic acid phenethyl ester 40 days before the grass carp hibernates. During the feeding period, the grass carp is fed three times a day in the morning, noon and afternoon respectively, and each feeding is one hour. This feeding method is not only easy to operate, but also can ensure that the grass carp fully absorbs the nutrients in the feed. Through practical verification, the application effect of the feed is remarkable, and the health status of the grass carp during the hibernation period can be significantly improved, and the fat loss rate can be reduced, which provides strong technical support for the development of the grass carp breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments are briefly described below.

[0020] Figure 1 Effects of feeding grass carp with a high-fat diet containing caffeic acid phenethyl ester 40 days before wintering on weight loss and fat loss rate during the wintering; (A) feeding time and wintering time, (B) water temperature during the wintering, (C) fish weight at the beginning of wintering, (D) fish weight at the end of wintering; (E): weight loss of grass carp after the end of wintering; (F): fat loss rate of grass carp after the end of wintering.

[0021] Figure 2Effects of feeding grass carp a high-fat diet containing caffeic acid phenethyl ester 40 days before wintering on adipocyte proliferation and lipid accumulation in adipose tissue of grass carp; (A) HE staining of adipose tissue at the beginning of wintering, (B) HE staining of adipose tissue at the end of wintering, (C) abdominal fat index at the beginning of wintering, (D) abdominal fat index at the end of wintering, (E) change rate of abdominal fat index before and after wintering.

[0022] Figure 3 Effects of feeding grass carp with a high-fat diet containing caffeic acid phenethyl ester 40 days before wintering on liver lipid utilization during the wintering period; (A) HE staining of liver tissue at the beginning of wintering, (B) HE staining of liver tissue at the end of wintering, (C) liver triglyceride content at the beginning of wintering, (D) liver triglyceride content at the end of wintering, (E) liver triglyceride utilization during the wintering period.

[0023] Figure 4 Effects of feeding grass carp with a high-fat diet containing caffeic acid phenethyl ester 40 days before wintering on serum biochemistry of grass carp during wintering; (A) AST content, (B) ALT content, (C) HDL cholesterol content, (D) LDL cholesterol content, (E) triglyceride content, (F) interleukin-1β, (G) tumor necrosis factor-α. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] The invention provides a caffeic acid phenethyl ester high-fat feed for use in improving the health status of grass carp during winter and reducing the fat loss rate. 400 mg / kg to 1200 mg / kg of caffeic acid phenethyl ester is added to the grass carp high-fat feed.

[0026] Calculated by the total weight of feed, the high-fat feed for grass carp includes 20% soybean meal, 14% rapeseed meal, 14.5% cottonseed meal, 20% flour, 5% DDGS (dry distiller's grains and solubles), 10% rice bran, 8.1% soybean oil, 2% bentonite, 0.5% choline chloride, 2% monocalcium phosphate, 1% grass carp premix, 0.1% antioxidant, 2.76% rice husk powder, and 0.04%~0.12% caffeic acid phenethyl ester.

[0027] 40 days before wintering, grass carp were fed with high-fat feed containing caffeic acid phenethyl ester. During the feeding period, they were fed artificially at fullness, 3 times a day (9:00, 13:00, 17:00), and each feeding was 1 hour. The pond cage water quality was measured at 12 noon every day. During the breeding period, the ammonia nitrogen in the breeding pond water was less than 0.5 mg / L, the nitrite was less than 0.05 mg / L, the sulfide was less than 0.05 mg / L, the pH was 7-8, and the dissolved oxygen was 7-8 mg / L. After the feeding was completed, wintering began; The present invention provides a method for preparing the high-fat feed containing caffeic acid phenethyl ester, and the specific steps are as follows: (1) Accurately weigh caffeic acid phenethyl ester and an antioxidant, and mix them to obtain a mixture 1; (2) accurately weighing choline chloride and mixing it with mixture 1 to obtain mixture 2; (3) accurately weighing choline chloride and mixing it with mixture 2 to obtain mixture 3; (4) accurately weighing the grass carp premix and mixing it with mixture 3 to obtain mixture 4; (5) accurately weighing bentonite, calcium dihydrogen phosphate, and rice husk powder, and mixing them with mixture 4 to obtain mixture 5; (6) Weighing DDGS, rice bran, rapeseed meal, cottonseed meal, soybean meal, and flour in that order, and mixing them with mixture 5 to obtain mixture 6; (7) Accurately weighing soybean oil, and adding it to mixture 6 and mixing well to obtain mixture 7; (8) Weighing 20% ​​water by weight of the mixture 7, and adding the water to the mixture 7 and mixing well to obtain a mixture 8; (9) The mixture 8 was made into pellet feed using a ring die pelletizer with a diameter of 4 mm and stored at -20°C.

[0028] (10) Feeding the grass carp with the pellet feed prepared above 40 days before wintering can promote the proliferation of fat cells, thereby improving the health of the grass carp during wintering and reducing fat loss.

[0029] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention will be further described in detail with reference to the embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Example 1. Experimental Design The experiment designed five treatments, namely: normal fat level group (diet fat level was 5%, Control), high fat level group (diet fat level was 10%, HF), and the high fat level group supplemented with 400mg / kg (C400), 800mg / kg (C800) and 1200mg / kg (C1200). During the experiment, three parallels were set for each treatment.

[0031] 2. Experimental feed The experimental feed formula is shown in Table 1. All feed raw materials were purchased from Huaqin Agriculture and Animal Husbandry Technology Co., Ltd. (Shaanxi Province, China); caffeic acid phenethyl ester was purchased from MCE (Shanghai, China). All purchased solid feed particles were crushed by a grinder and passed through a 0.25 mm sieve. Then, they were fully mixed according to the feed formula to make pellet feed with a diameter of 4 mm. After natural air drying in a ventilated and dry place, they were placed in a -20 °C refrigerator for storage.

[0032] Table 1 Experimental feed formula (dry matter, %)

[0033] 3. Experimental grass carp and feeding management In order to allow the experimental fish to adapt to the breeding environment in advance, all experimental grass carps were temporarily acclimated in pond cages for one month before formal feeding. During the temporary acclimation period of the experimental fish, commercial feed (Tongwei) with the same protein and fat levels as the experimental feed was used. On September 21, 2023, after all the experimental fish were starved for 24 hours, grass carps of uniform size and health were selected and placed in 15 cages, with 20 fish in each cage, and the average initial weight was 520.74±0.76 g. From September 21 to October 31, 2023, the experimental fish were fed with the prepared experimental feed for 40 days before wintering. During the feeding period, they were fed artificially and fed 3 times a day (9:00, 13:00, 17:00), each time for 1 hour. The water quality of the pond cages was measured at 12 noon every day. During the experimental breeding period, ammonia nitrogen was less than 0.5 mg / L, nitrite was less than 0.05 mg / L, sulfide was less than 0.05 mg / L, pH was 7-8, and dissolved oxygen was 7-8 mg / L. After the feeding was completed, feeding was stopped on November 1, 2023, and a 154-day wintering period began. The average water temperature during the wintering period was 11.8℃.

[0034] 4. Experimental sampling Sampling and analysis were performed after the feeding was completed (i.e., at the beginning and after the wintering). After the feeding was completed, all experimental fish were starved for 24 hours and then fished out and weighed. Then, 6 fish were randomly selected for sampling, and the remaining fish were returned to the original cage for wintering. During the sampling process, the 6 fish were weighed one by one, and then dissected one by one to obtain adipose tissue and liver tissue and weigh them. Then, part of the obtained adipose and liver tissue was stored in a refrigerator at -80 ℃, and part was used for histopathological section observation.

[0035] After the wintering, all experimental fish were fished out and weighed. Then, 6 fish were randomly selected for sampling, and the remaining fish were returned to the original cage for wintering. During the sampling process, blood was drawn from the vein with a syringe, centrifuged at 3500 r / min for 15 min to obtain serum, which was quickly frozen in liquid nitrogen and stored in a refrigerator at -80 ℃ for the determination of serum biochemical indicators. Then, adipose tissue and liver tissue were obtained and weighed, and then part of the obtained adipose and liver tissue was stored in a refrigerator at -80 ℃, and part was used for histopathological section observation.

[0036] 5. Sample determination 5.1 Calculation of biological traits Intraperitoneal fat index (%) = 100 × abdominal fat weight / body weight Body weight loss rate (%) = 100 × (weight before wintering - weight after wintering) / weight before wintering 5.2 Tissue sections The tissue samples fixed in 4% paraformaldehyde were commissioned to Chengdu Lilai Biotechnology Co., Ltd. (Chengdu, China) for tissue sectioning (paraffin sectioning, HE staining, Oil Red O staining). The prepared sections were photographed under a Nikon SMZ18 research-grade upright microscope (10× eyepiece, 20× objective lens, magnification 200 times) at the College of Veterinary Medicine, Northwest Agriculture and Forestry University.

[0037] 5.3 Blood biochemical indicators Serum samples were tested by Chengdu Lilai Biotechnology Co., Ltd. (Chengdu, China), including alanine aminotransferase (ALT, U / L), aspartate aminotransferase (AST, U / L), triglycerides (TG, mmol / L), low-density lipoprotein cholesterol (LDL-C, mmol / L) and high-density lipoprotein cholesterol (HDL-C, mmol / L), interleukin-1β (IL-1β, ng / L), and tumor necrosis factor-α (TNF-α, ng / L).

[0038] 5.4 Detection of triglycerides To detect the triglyceride (TG) content, the present invention used a tissue and cell triglyceride content detection kit (E1013, Beijing Pulilai Gene Technology Co., Ltd., Beijing, China).

[0039] 5.5 Data Statistical Analysis All experimental data were analyzed by one-way ANOVA (Shapiro-Wilk normality test) and Levene homogeneity of variance test using SPSS 19.0 for Windows (SPSS, IL, USA). P <0.05 indicated significant difference). When the difference was significant, Duncan's multiply range test was performed on the means of each treatment group. All data were expressed as mean ± standard error (mean ± SE).

[0040] 6. Application Effects 6.1 Feeding grass carp with a high-fat diet containing caffeic acid phenethyl ester 40 days before wintering promoted the proliferation of grass carp adipocytes like Figure 2 As shown in the figure, within 40 days before wintering, grass carp ingested a high-fat diet containing caffeic acid phenethyl ester, which significantly promoted the proliferation of fat cells in grass carp. That is, the fat cell area of ​​the treatment groups with caffeic acid phenethyl ester (HF+C400, HF+C800, HF+C1200) was significantly lower than that of the HF group ( Figure 2 (A)), and the hyperplastic state of adipocytes is maintained until the end of hibernation ( Figure 2 (B)). Figure 2 (A): HE staining of grass carp fat tissue after feeding before wintering; Figure 2 (B): HE staining of grass carp adipose tissue after overwintering. Figure 2 (C): Abdominal fat index of grass carp after feeding before wintering; Figure 2 (D): Abdominal fat index of grass carp after wintering; Figure 2 (E): Loss rate of abdominal fat tissue of grass carp during wintering.

[0041] 6.2 Feeding grass carp with a high-fat diet containing caffeic acid phenylethyl ester 40 days before wintering reduced the weight loss and fat loss rate of grass carp after wintering like Figure 1 As shown in Figure 2, within 40 days before wintering, grass carp fed a high-fat diet containing caffeic acid phenethyl ester had no difference in body weight between the control group and the high-fat treatment group ( P >0.05) ( Figure 1(C)), but after the winter, the weight of grass carp fed with high-fat diet containing caffeic acid phenethyl ester was significantly higher than that of the control group and high-fat treatment group ( P <0.05)( Figure 1 (D)), and weight loss ( Figure 1 (E)) and the lowest fat loss rate ( P <0.05)( Figure 1 (F) The fat loss rates of the control group and the high-fat level treatment group during the winter were 14.67% and 11.19%, respectively, while the average fat loss rate of the group treated with caffeic acid phenethyl ester was only 7.20%. Compared with the control group and the high-fat level treatment group, the fat loss rate of grass carp in the group treated with caffeic acid phenethyl ester during the winter was reduced by 7.47% and 3.99%, respectively; Figure 1 (A): Time nodes and schematic diagram of the feeding experiment; Figure 1 (B): Graph of water temperature changes during the feeding experiment. Figure 1 (C): Weight of grass carp after 40 days of feeding before wintering; Figure 1 (D): Weight of grass carp after wintering; Figure 1 (E): Weight loss of grass carp after wintering; Figure 1 (F): The fat loss rate of grass carp after wintering. 6.3 Feeding grass carp with a high-fat diet containing caffeic acid phenethyl ester 40 days before wintering improves the energy utilization pattern of grass carp during wintering like Figure 2 and Figure 3 As shown in Figure 2, after grass carp ingested a high-fat diet containing caffeic acid phenethyl ester before wintering, the loss rate of abdominal fat tissue after wintering was significantly higher than that of the high-fat group (HF) ( Figure 2 E); liver triglyceride content after wintering ( Figure 3 B) Triglyceride utilization rate in the liver during wintering ( Figure 3 D) were significantly higher and lower than the high-fat group (HF), indicating that the energy required for life activities during the winter mainly comes from adipose tissue, thereby reducing the energy supply of liver tissue, thereby reducing the burden of grass carp liver tissue during the winter, and ultimately improving the health of grass carp liver tissue. Note: Figure 3 (A): Oil red staining of grass carp liver after the feeding experiment; Figure 3 (B): Oil red staining of grass carp liver after wintering. Figure 3 (C): Triglyceride content in grass carp liver after feeding before wintering; Figure 3 (D): Triglyceride content in liver of grass carp fed after wintering; Figure 3 (E): Utilization of triglycerides in grass carp liver during wintering.

[0042] 6.4 Feeding grass carp with a high-fat diet containing caffeic acid phenethyl ester 40 days before wintering improves the health of grass carp liver during wintering like Figure 4 As shown in the figure, after grass carp ingested a high-fat diet containing caffeic acid phenethyl ester before wintering, its serum aspartate aminotransferase, alanine aminotransferase, low-density lipoprotein cholesterol, and triglyceride levels were significantly reduced after wintering, while interleukin-1β, tumor necrosis factor-α, and high-density lipoprotein cholesterol levels were significantly increased ( P <0.05), thus improving the liver health of grass carp during the winter. Figure 4 (A)-(G) are the serum aspartate aminotransferase content, alanine aminotransferase content, high-density lipoprotein cholesterol content, low-density lipoprotein cholesterol content, triglyceride content, interleukin-1β content, and tumor necrosis factor-α content of grass carp serum after the end of wintering.

[0043] Combined with the above results, the present invention can not only improve the health of grass carp during the winter, but also reduce the weight loss and fat loss rate of grass carp during the winter. While reducing breeding losses, the present invention also provides guarantees for the safe wintering of grass carp and the rapid recovery of physical fitness after wintering. In summary, the present invention can be used as an indispensable nutrition improvement strategy to ensure the healthy breeding of grass carp.

Claims

1. Application of caffeic acid phenethyl ester in fish feed.

2. The application according to claim 1, characterized in that: The fish is grass carp.

3. A high-fat feed, characterized in that: The high-fat feed contains caffeic acid phenethyl ester.

4. A high-fat feed according to claim 3, characterized in that: The added amount of the caffeic acid phenethyl ester is 400 mg / kg to 1200 mg / kg.

5. A method for improving the health of grass carp during winter and reducing its fat loss rate, characterized in that: The grass carp is fed with the high-fat feed according to claim 3 or 4 40 days before wintering.

6. A method for improving the health of grass carp during winter and reducing fat loss rate according to claim 5, characterized in that: During the feeding period, the fish were fed artificially to satiety, three times a day at 9:00, 13:00, and 17:00, each feeding lasting 1 hour.

7. A high-fat feed for grass carp, characterized in that: The grass carp high-fat feed contains caffeic acid phenethyl ester.

8. The high-fat feed for grass carp according to claim 7, characterized in that: The added amount of the caffeic acid phenethyl ester is 400 mg / kg to 1200 mg / kg.

9. Use of the high-fat feed for grass carp according to claim 7 or 8 in improving the health of grass carp during winter and reducing its fat loss rate.

10. The use according to claim 9, characterized in that: The grass carp are fed with the high-fat feed 40 days before wintering. During the feeding period, the grass carp are fed artificially to satiety, three times a day at 9:00, 13:00, and 17:00, each time for 1 hour.

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