CN109965148B - Application of mulberry leaf extract in inhibiting freshwater fish culture stress, feed and feed preparation method - Google Patents

Abstract

The invention provides an application of mulberry leaf extract in inhibiting freshwater fish culture stress, a feed and a feed preparation method. The preparation method of the mulberry leaf extract comprises the following steps: oven drying folium Mori, pulverizing, extracting with cyclohexane, filtering, extracting with diethyl ether, filtering, and distilling the filtrate under reduced pressure to constant weight. The invention also provides the proportion of the mulberry leaf extract in the anti-stress feed and a preparation method of the feed. The mulberry leaf extract is used as an anti-stress agent in the feed, and can inhibit stress reaction of freshwater fish in cultivation caused by high density, copper sulfate, trichlorfon and the like, so that the anti-stress capability of the freshwater fish is improved, the morbidity and mortality of the freshwater fish are reduced, meanwhile, the novel use of mulberry leaves is developed, and the mulberry She Ziyuan is effectively utilized.

Description

Application of mulberry leaf extract in inhibiting freshwater fish culture stress, feed and feed preparation method

Technical Field

The invention belongs to the technical field of fish culture, and particularly relates to application of a mulberry leaf extract in inhibiting freshwater fish culture stress, a feed and a feed preparation method.

Background

High density intensification is an important feature of current aquaculture. The high-density cultivation improves the feeding amount, increases the cultivation waste in the water body, worsens the water quality, and is characterized in that the dissolved oxygen amount in the water is reduced and the ammonia nitrogen and nitrite content exceeds the standard. It has been reported that: these factors can cause oxidative stress damage to the fish, thereby reducing digestion and absorption of the fish to the feed, reducing growth and immune function of the fish, and increasing morbidity and mortality of the fish. High density farming also results in the over-use of chemical disinfectants and pesticides. Copper sulfate is one of the most commonly used metal disinfectant pesticides in aquaculture, and its effective use concentration in water is typically 0.5-1.0 mg/L. Studies have demonstrated that the use of copper sulfate in aquaculture water induces oxidative stress in fish and thereby reduces the ingestion, digestion, absorption, growth and immune function of fish. Trichlorfon is one of the most commonly used organophosphorus pesticides in aquaculture, and is a cholinesterase inhibitor. Trichlorfon poisoning can cause muscle dysfunction in animals and loss of balance. It has been reported that trichlorfon can induce oxidative stress in farmed fish and adversely affect the digestive absorption, respiration and immune functions of fish. When fish is stimulated by the stress factors, the levels of the auxiliary renal cortex are hypertrophic, the in vivo glucocorticoids, the steroid corticoids and the like are increased, so that the activities of blood lymphocytes, macrophages, neutrophils and lysozyme are drastically reduced, the immune system of animals is inhibited, the weight gain is reduced, the death rate is increased, and the economic benefit and the ecological benefit of cultivation are seriously influenced.

The mulberry is planted in China for a long time, and the mulberry leaves are mainly used for silkworm breeding and can also be eaten and used as medicines. The modern pharmaceutical research results show that the mulberry leaves have various pharmacological activities and have the functions of removing in-vitro and in-vivo Reactive Oxygen Species (ROS) and chelating metal ions, but the application of Guan Sangshe extract in the aspect of inhibiting freshwater fish culture stress is not reported at present. The mulberry leaves in China are rich in resources, but the utilization is very limited, and a large amount of mulberry leaves are abandoned each year. The application of Sang Shexin is developed, the traditional mulberry leaf utilization structure is improved, the comprehensive utilization potential of the mulberry leaves is exerted, waste is changed into valuable, and the economic and social benefits of the mulberry leaves are improved, so that the method has very important significance.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application of mulberry leaf extract in inhibiting freshwater fish culture stress, a feed and a feed preparation method. The mulberry leaf extract is used as an anti-stress agent in the feed, so that the culture stress reaction of the freshwater fish can be inhibited, the influence of stress factors such as high-density culture, copper sulfate, trichlorfon and the like can be reduced, the anti-stress capability of the freshwater fish can be improved, the morbidity and mortality of the freshwater fish can be reduced, the novel use of the mulberry leaf can be developed, and the mulberry She Ziyuan can be effectively utilized.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: the invention provides an application of mulberry leaf extract in inhibiting freshwater fish culture stress.

Further, the mulberry leaf extract is added into the feed for use, and the adding proportion is 4.52-7.00 wt per mill.

Further, the folium Mori extract is folium Mori diethyl ether extract, sang Shehuan hexane extract or folium Mori acetone extract.

Further, sang Shehuan hexane extract is prepared by the following method: drying and crushing mulberry leaves, and mixing mulberry leaf powder and cyclohexane according to a ratio of 1: mixing 5-8 proportion, stirring, extracting, filtering, repeating extracting for 2-3 times to obtain filtrate and first filter residue, mixing the filtrates, and distilling under reduced pressure to constant weight to obtain the final product;

the mulberry leaf diethyl ether extract is prepared by the following method: mixing the first filter residue obtained in the extraction process of Sang Shehuan hexane extract with diethyl ether according to a ratio of 1: mixing 5-8 proportion, stirring, extracting, filtering, repeating extracting for 2-3 times to obtain residue-free filtrate and second filter residue, mixing the filtrates, and distilling under reduced pressure to constant weight to obtain the final product;

the mulberry leaf acetone extract is prepared by the following method: mixing the second filter residue obtained in the extraction process of the mulberry leaf diethyl ether extract with acetone according to a ratio of 1: mixing 5-8 proportion, stirring, extracting, filtering, repeating extracting for 2-3 times to obtain residue-free filtrate and third filter residue, mixing the filtrates, and vacuum distilling to constant weight.

An anti-stress agent for freshwater fish comprises the above folium Mori extract.

A feed comprises basic feed and the above folium Mori extract.

Further, the mulberry leaf extract accounts for 4.52 to 7.00 per mill of the total weight of the feed.

Further, the mulberry leaf extract accounts for 4.90 per mill of the total weight of the feed.

Further, the basic feed comprises the following components in parts by weight: 23.00 to 25.00 portions of fish meal, 31.00 to 33.00 portions of bean pulp, 0.60 to 0.80 portion of DL-methionine, 36.00 to 38.00 portions of flour, 3.00 to 3.50 portions of grease, 0.80 to 1.30 portions of vitamin additive and 0.80 to 1.30 portions of mineral additive.

Wherein the vitamin additive comprises the following components in parts by weight: retinol 0.80 parts, vitamin D ₃ 0.48 part of DL-alpha-tocopherol 20.00 parts and vitamin K ₃ 0.43 part of vitamin B ₁ 0.11 part, 0.63 part of riboflavin, 0.92 part of pyridoxine, 0.10 part of cyanocobalamin, 2.73 parts of D-calcium pantothenate, 2.82 parts of nicotinic acid, 5.00 parts of D-biotin, 52.33 parts of inositol and 0.52 part of folic acid.

The mineral additive comprises the following components in parts by weight: feSO ₄ ·7H ₂ O69.70 parts, cuSO ₄ ·5H ₂ O1.20 parts, znSO ₄ ·7H ₂ O21.64 parts, mnSO ₄ ·H ₂ O4.09 parts, na ₂ SeO ₃ ·5H ₂ O2.50 parts and KI 2.90 parts.

The oil is soybean oil, corn oil, rapeseed oil, peanut oil or mixture of sunflower seed oil and fish oil.

The preparation method of the feed comprises the following steps:

dissolving folium Mori extract in oil, mixing with other components of basic feed, and granulating.

In summary, the invention has the following advantages:

1. according to the invention, the mulberry leaf extract is added into the fish feed as an anti-stress agent, so that the stress response of high-density cultivation, copper sulfate, trichlorfon and the like on the freshwater fish is inhibited, the anti-stress capability of the freshwater fish is improved, the side turnover of the fish body is inhibited, the food intake and weight gain of the freshwater fish are recovered, and the economic benefit of freshwater fish cultivation is further improved.

2. The invention determines that the optimal addition amount of the mulberry leaf diethyl ether extract in the anti-stress feed is 4.90g/kg. Wherein, the concentrations of the mulberry leaf diethyl ether extract with high anti-culture density and optimal anti-cupric sulfate and anti-dipterex stress effects are respectively 4.85g/kg, 4.52g/kg and 4.90g/kg of feed.

3. The invention provides an extraction method of mulberry leaf extract, which adopts two steps of extraction, firstly uses cyclohexane for primary extraction, and then uses diethyl ether for further extraction to obtain mulberry leaf diethyl ether extract; greatly reduces cyclohexane extraction components in the mulberry leaf diethyl ether extract, and has high purity of extracted active components and good extraction effect.

4. The invention determines that the active ingredient of the mulberry leaf extract for resisting freshwater fish culture stress is a flavonoid compound. The effects of chelating metal ions (MCA) of mulberry leaf extract, recovering freshwater fish ingestion, improving fish intestinal anti-superoxide anion activity (ASA) and anti-hydroxyl radical Activity (AHR) and reducing fish intestinal Malondialdehyde (MDA) content are closely related to the flavonoid content. The mechanism of the mulberry leaf extract for resisting high-density stress is that the mulberry leaf extract improves the antioxidant function of organisms. Wherein, the flavonoids content of the mulberry leaf diethyl ether extract is 61.68-65.70 g/kg.

5. The invention provides a method for adding mulberry leaf extract as an anti-stress agent of freshwater fish into feed, namely, the mulberry leaf extract is dissolved into grease and then mixed with other raw materials for granulating to obtain the feed for the fish; the redundant mulberry She Ziyuan is changed into valuable, so that the rich mulberry She Ziyuan is effectively utilized, the purpose of the mulberry is enriched, and the economic benefit is improved.

Drawings

FIG. 1 is a graph showing in vitro chelating metal ion activity (MCA) of mulberry leaf extracts with different polarities and feeding recovery rate of crucian;

FIG. 2 is a graph showing the relationship between flavonoid content of mulberry leaf extracts with different polarities and chelated metal ion activity (MCA) and feeding recovery rate of crucian;

FIG. 3 is a schematic diagram showing the effect of culture density on crucian weight gain;

FIG. 4 is a schematic diagram showing the effect of culture density on crucian feed efficiency;

FIG. 5 is a schematic diagram showing the effect of culture density on mortality of crucian;

FIG. 6 is a schematic representation of the content of superoxide anion activity (ASA), hydroxyl radical Activity (AHR) and Malondialdehyde (MDA) in the intestine of crucian fed with mulberry leaf extract of different polarity;

FIG. 7 is a graph showing the relationship between flavonoid content of each extract of mulberry leaves and the content of superoxide anion activity (ASA), hydroxyl radical Activity (AHR) and Malondialdehyde (MDA) in the intestinal tract of crucian carp;

FIG. 8 is a schematic diagram of broken line regression analysis of the recovery rate of weight gain of crucian carp after feeding the mulberry leaf diethyl ether extract for 60 days;

FIG. 9 is a schematic diagram showing the effect of different copper sulfate contents in water on crucian feeding rate;

FIG. 10 is a schematic diagram showing the effect of different copper sulfate content in water on mortality of crucian carp;

FIG. 11 is a schematic diagram showing the effect of different trichlorfon content in water on the rollover rate of crucian;

FIG. 12 is a schematic illustration of the effect of different trichlorfon levels in water on mortality of crucian carp;

FIG. 13 is a schematic diagram of a broken line regression analysis of the feeding recovery rate of crucian carp after copper sulfate stress;

fig. 14 is a schematic diagram of a broken line regression analysis of the rollover inhibition rate of crucian carp after dipterex stress.

Detailed Description

Example 1

The preparation method of the mulberry leaf extract comprises the following steps:

(1) Drying 1000g of mulberry leaves in a blast drying oven at 50 ℃ to constant weight, crushing and sieving with a 18-mesh sieve to obtain mulberry leaf powder;

(2) 400g of mulberry leaf powder is taken and mixed with cyclohexane according to the proportion of 1:6g/mL, and stirred for 6 hours at 600r/min, and clear filtrate and filter residue are obtained after filtration;

(3) Repeating the step (2) for 2 times, mixing the filtrates obtained in the step (3), distilling the filtrate under reduced pressure to constant weight, and removing cyclohexane to obtain Sang Shehuan hexane extract.

Example 2

The preparation method of the mulberry leaf extract comprises the following steps:

(1) Drying 1000g of mulberry leaves in a blast drying oven at 50 ℃ to constant weight, crushing and sieving with a 18-mesh sieve to obtain mulberry leaf powder;

(2) 400g of mulberry leaf powder is taken, mixed according to the proportion of 1:6g/mL of mulberry leaf powder and cyclohexane, stirred for 6 hours at 600r/min, filtered to obtain filter residues, and the filter residues are repeatedly extracted for 2 times;

(3) Mixing the filter residue obtained in the step (2) with diethyl ether according to the proportion of 1:6g/mL, stirring for 6h at 600r/min, filtering to obtain clear filtrate and filter residue, repeatedly extracting the filter residue for 2 times, and combining the filtrates extracted for 3 times;

(4) Distilling the filtrate obtained in the step (3) under reduced pressure to constant weight to remove diethyl ether, thereby obtaining the mulberry leaf diethyl ether extract.

Example 3

The preparation method of the mulberry leaf extract comprises the following steps:

(1) Drying 1000g of mulberry leaves in a blast drying oven at 50 ℃ to constant weight, crushing and sieving with a 18-mesh sieve to obtain mulberry leaf powder;

(2) 400g of mulberry leaf powder is taken, mixed according to the proportion of 1:6g/mL of mulberry leaf powder and cyclohexane, stirred for 6 hours at 600r/min, filtered to obtain filter residues, and the filter residues are repeatedly extracted for 2 times;

(3) Mixing the filter residue obtained in the step (2) with diethyl ether according to the proportion of 1:6g/mL, stirring for 6 hours at 600r/min, filtering to obtain filter residue, and repeatedly extracting the filter residue for 2 times;

(4) Mixing the filter residue obtained in the step (3) with acetone according to the ratio of 1:6g/mL, stirring for 6h at 600r/min, filtering the mixed solution to obtain clear residue-free filtrate and filter residue, repeatedly extracting the filter residue for 2 times, and combining the filtrates extracted for 3 times;

(5) Distilling the filtrate obtained in the step (4) under reduced pressure to constant weight to remove acetone and obtain the mulberry leaf acetone extract.

Example 4

The preparation method of the mulberry leaf extract comprises the following steps:

(1) Drying 1000g of mulberry leaves in a blast drying oven at 50 ℃ to constant weight, crushing and sieving with a 18-mesh sieve to obtain mulberry leaf powder;

(2) 400g of mulberry leaf powder is taken, mixed according to the proportion of 1:6g/mL of mulberry leaf powder and cyclohexane, stirred for 6 hours at 600r/min, filtered to obtain filter residues, and the filter residues are repeatedly extracted for 2 times;

(3) Mixing the filter residue obtained in the step (2) with diethyl ether according to the proportion of 1:6g/mL, stirring for 6 hours at 600r/min, filtering to obtain the filter residue, and repeatedly extracting the filter residue for 2 times;

(4) Mixing the filter residue obtained in the step (3) with acetone according to the ratio of 1:6g/mL, stirring for 6 hours at 600r/min, filtering to obtain the filter residue, and repeatedly extracting the filter residue for 2 times;

(5) Mixing the filter residue obtained in the step (4) with water according to the proportion of 1:6g/mL, stirring for 6 hours at 600r/min, centrifuging to obtain clear filtrate and filter residue, repeatedly extracting the filter residue for 2 times, and combining the filtrates extracted for 3 times;

(6) Distilling the filtrate obtained in the step (5) under reduced pressure to constant weight, and removing water to obtain the mulberry leaf water extract.

The flavonoid compounds and the chelated metal ions (Fe) of the four mulberry leaf extracts obtained in examples 1 to 4 were measured by a chemical method ²⁺ ) Activity (MCA), the results of which are shown in Table 1 and FIG. 1. Meanwhile, the correlation analysis of MCA and flavonoid content of four mulberry leaf extracts is carried out, and the analysis result is shown in figure 2. The data in table 1 and figure 1 are expressed as the mean ± standard deviation of 3 replicates; within the same index, the data shoulder letters differ to indicate significant differences between the data, and the same indicates insignificant differences (P<0.05). The data in figure 2 are presented as an average of 3 replicates.

TABLE 1 flavonoid content in mulberry leaf extracts with different polarities

As shown in Table 1, the flavonoids content of the mulberry leaf diethyl ether extract is highest and is 61.68-65.70 g/kg. As can be seen from FIG. 1, the mulberry leaf extract has a function of chelating the transition metal ion, and among them, the diethyl ether extract of mulberry leaf works best. As can be seen from FIG. 2, the MCA of the mulberry leaf extract has a close relationship with the flavonoid content thereof. Therefore, flavonoids in the mulberry leaf extract sequester excessive metal ions.

Example 5

After a plurality of crucian fries are purchased locally and temporarily cultured in a culture room for one week, 720 tails of the crucian fries with the weight of 8.0+/-0.2 g are selected and randomly divided into 8 treatment groups of 4 fish tanks, wherein the number of the tails of the 8 treatment groups is respectively as follows: 5. 10, 15, 20, 25, 30, 35 and 40 tails/cylinders. Wherein, the shape, the size and the color of each fish tank are the same, the size is 30cm multiplied by 40cm, and one 800L/h flow aerator is arranged in each fish tank. Tap water is used as a water source, and the volume of water in the tank is 31L. Controlling the culture room temperature to be 22+/-2 ℃; oxygen is continuously supplied by the aerator; feeding fish meal, bean pulp and omnivorous fish feed; changing water every 2-3 days, and the cultivation period is 60 days. After a 60-day cultivation period, the weight gain, feeding amount, feed efficiency and mortality of each treated group of fish were examined, the influence of the cultivation density on the weight and feeding amount of crucian were shown in table 2, and the influence of the cultivation density on the weight gain, feed efficiency and mortality of the fish were shown in fig. 3 to 5, respectively. Wherein, the data in Table 2 and FIGS. 3-5 are expressed as the mean.+ -. Standard deviation of 4 replicates; in the same index data, the different superscript letters indicate that the difference between the data is significant, and the same letter indicates that the difference is not significant (P < 0.05). Weight gain = last-first weight; feed efficiency = 100 x weight gain/food intake.

TABLE 2 influence of cultivation Density on Carassius auratus weight and food intake

As shown in Table 2, with increasing cultivation density, the feed intake tended to increase and decrease, and the cultivation density at which the maximum feed intake was 0.32 to 0.48 tail/L water. As shown in FIG. 3, the weight gain gradually decreased with increasing cultivation density, and the cultivation density with the least weight gain was 0.97-1.29 tail/L water. As is clear from FIG. 4, the feed efficiency gradually decreased with increasing cultivation density, and the density of the lowest feed efficiency was 0.97 to 1.29 tail/L water, and the highest feed efficiency was 0.16 to 0.48 tail/L water. As is clear from FIG. 5, the fish starts to die when the cultivation density is 1.13 to 1.29 tail/L water.

As can be seen from the above, the optimum cultivation density was 0.48 tail/L water, and the cultivation density, which produced the greatest density stress without causing death of fish, was 0.97 tail/L water.

Example 6

An anti-stress feed comprises the following components in parts by weight: 24.00 parts of fish meal, 32.00 parts of soybean meal, 0.70 part of DL-methionine, 37.00 parts of flour, 1.50 parts of fish oil, 1.80 parts of sunflower seed oil, 0.49 part of mulberry leaf extract, 1.00 parts of vitamin additive and 1.00 parts of mineral additive.

Wherein the vitamin additive comprises the following components in parts by weight: retinol 0.80 parts, vitamin D ₃ 0.48 part of DL-alpha-tocopherol 20.00 parts,Vitamin K ₃ 0.43 part of vitamin B ₁ 0.11 part, 0.63 part of riboflavin, 0.92 part of pyridoxine, 0.10 part of cyanocobalamin, 2.73 parts of D-calcium pantothenate, 2.82 parts of nicotinic acid, 5.00 parts of D-biotin, 52.33 parts of inositol and 0.52 part of folic acid.

The mineral additive comprises the following components in parts by weight: feSO ₄ ·7H ₂ O69.70 parts, cuSO ₄ ·5H ₂ O1.20 parts, znSO ₄ ·7H ₂ O21.64 parts, mnSO ₄ ·H ₂ O4.09 parts, na ₂ SeO ₃ ·5H ₂ O2.50 parts and KI 2.90 parts.

The preparation method of the anti-stress feed comprises the following steps:

(1) Mixing the mulberry leaf extract with sunflower seed oil and fish oil according to a feed formula, and carrying out ultrasonic oscillation for 40min to uniformly mix the mulberry leaf extract;

(2) And (3) adding the rest component raw materials into the mixture obtained in the step (1), uniformly mixing, and preparing the granular feed with the diameter of 2 mm.

Example 7

The 4 extracts of mulberry leaves prepared in examples 1 to 4 were added to feeds at concentrations of 0 and 2g/kg, and corresponding 5 pellet feeds were prepared according to the formulation and preparation method in example 6.

Under the conditions of optimal stress density (0.97 tail/L water) and the same cultivation environment as in example 5, the 495 tail of the crucian fry with the weight of 23.3+/-1.0 g is selected and randomly divided into 6 treatment groups, and 3 fish tanks are arranged in each group. The fish tail numbers of the 6 treatment groups are respectively as follows: 15. 30, 30 and 30 tails/jar, named control, induction, sang Shehuan hexane extract, mulberry leaf diethyl ether extract, mulberry leaf acetone extract and mulberry leaf water extract group, respectively. The 6 treatment groups were fed with feeds containing 0, 0 and 2g/kg Sang Shehuan hexane extract, mulberry leaf diethyl ether extract, mulberry leaf acetone extract and mulberry leaf water extract, respectively. Feeding 4 times per day, 8 in the morning: feeding 00 for the first time, and feeding every 4 hours later, 8:00 is fed for the last time. Feeding 0.5 g/jar each time on day 1; later, 0.1 g/cylinder is added for each feeding; and the feeding amount is not increased until the surplus material appears. The residual materials are ensured every day later, the residual baits are quickly fished out after 30 minutes after each feeding, and the residual baits are timely dried and the weight is recorded; and after the residual materials disappear, the feeding amount is increased. After 10 days of feeding experiment, the feed intake and the fish weight of each treatment group were counted, and the feed intake rate was calculated. After 1 day of stop feeding, intestinal samples of fish were collected, and the content of superoxide anion resistant (ASA), hydroxyl radical resistant (AHR) and Malondialdehyde (MDA) in intestinal tissues of 6 treatment groups was determined by a kit (institute of biotechnology of built in south ky) method.

The results are shown in FIGS. 1 and 6, where data are expressed as the mean.+ -. Standard deviation of 3 replicates; within the same index, data shoulder letters differ to indicate significant differences between data, and the same indicates insignificant differences (P < 0.05). The figure shows that the mulberry leaf extract restores the feeding rate of crucian and the intestinal antioxidant activity of the crucian; the diethyl ether extract of mulberry leaves is the best. The correlation analysis results are shown in figures 2 and 7, where the data is presented as an average of 3 replicates. From the figure, the feeding recovery and antioxidation effects of the four extracts of the mulberry leaves are closely related to the flavonoid content of the mulberry leaves; it restores feeding function due to its chelating metal ions and improving intestinal antioxidant capacity.

Example 8

The mulberry leaf diethyl ether extract prepared in example 2 was added to feeds at concentrations of 0, 1, 2, 3, 4, 5, 6 and 7g/kg, respectively, and corresponding feeds were prepared according to the formulation and preparation method in example 6. Under the same cultivation environment with the optimal stress density of 0.97 tail/L water and the same cultivation environment as in the example 5, 7.2+/-0.2 g of crucian fry 1020 tail is selected and randomly divided into 9 treatment groups, and 4 fish tanks are arranged in each group. The fish tail numbers of the 9 treatment groups were respectively: 15. 30, 30 and 30 tails/cylinder, named control (D), induction (Y), 1g (E ₁ )、2g(E ₂ )、3g(E ₃ )、4g(E ₄ )、5g(E ₅ )、6g(E ₆ ) And 7g (E) ₇ ) Groups, 9 treatment groups were fed with feeds containing 0, 1, 2, 3, 4, 5, 6 and 7g/kg of mulberry leaf diethyl ether extract, respectively, for 60 days. After 60 days, the growth, feeding amount and feed efficiency of crucian carp were measured and the weight gain recovery rate was subjected to a broken line regression analysis, the results of which are shown in table 3 and fig. 8. The data in table 3 are expressed as mean ± standard deviation of 4 replicates; within the same index, data shoulder markThe letter difference indicates that the difference between the data is significant, and the same indicates that the difference is not significant (P<0.05). The data in figure 8 are presented as an average of 4 replicates. Weight gain recovery (%) = (E-Y)/(D-Y) ×100 (table 3).

TABLE 3 Effect of feeding a feed containing Mulberry leaf Ether extract (EEE) on high Density stress in crucian carp

As can be seen from Table 3, feeding the feed containing the mulberry leaf diethyl ether extract restored the growth, feeding amount and feed efficiency of the fish. As shown in FIG. 8, the optimum additive concentration of the mulberry leaf diethyl ether extract for resisting the high-density stress of cultivation is 4.85g/kg of feed.

Example 9

Under the same cultivation environment as in example 5, the weight of 21.2+/-1.1 g of the 210 tails of the crucian fries are selected and randomly divided into 7 treatment groups of 3 fish tanks, and 10 tails of fish are in each tank. Copper sulfate is dissolved in the water body of the fish tank of 7 treatment groups, the concentrations of the copper sulfate are respectively 0.0, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9mg/L, the 7 treatment groups are fed with fish meal soybean meal type omnivorous fish feed, the feed intake and the death rate of each treatment group are examined after four days, and the experimental results are shown in figures 9 and 10. Wherein, the data in the graph are expressed as the mean ± standard deviation of 3 replicates; the different numbers on the column indicate significant differences between the data, the same numbers indicate insignificant differences (P < 0.05).

As can be seen from fig. 9 and 10, the fish feed intake gradually decreases with increasing Cu concentration; when the Cu concentration is increased to 0.7mg/L water, the fish feeding amount is reduced to the minimum level; when Cu concentration is further increased to 0.8mg/L, death of fish occurs. Meanwhile, the Cu concentration that resulted in the maximum copper sulfate stress without causing death was 0.7mg/L.

Example 10

Under the same cultivation environment as in example 5, the same weight of the crucian fries 210 tails are selected and randomly divided into 7 treatment groups of 3 fish tanks each, and 10 tails per tank. Trichlorfon is dissolved in 7 treatment groups of fish tank water respectively to make the concentration of trichlorfon reach 0.0, 0.4, 1.0, 1.6, 2.2, 2.8 and 3.4mg/L. Four days later, the fish rollover rate and mortality rate of each treatment group were examined, and the results are shown in fig. 11 and 12. Wherein, the data in the graph are expressed as the mean ± standard deviation of 3 replicates; the different numbers on the column indicate significant differences between the data, the same numbers indicate insignificant differences (P < 0.05).

As can be seen from fig. 11 and 12, as the dipterex concentration increases, the fish turn over rate gradually increases, and when the dipterex concentration increases to 2.2mg/L, the fish turn over rate reaches the highest level, and when the dipterex concentration further increases to 2.8mg/L, the fish dies. The concentration that caused the greatest dipterex stress without causing fish death was 2.2mg/L water.

Example 11

Under the same cultivation environment as in example 5, 480 fish fries with a weight of 10.5+/-0.3 g are selected and randomly divided into 8 treatment groups, wherein each group comprises 4 fish tanks and 15 fish fries per tank. 8 treatment groups were fed with 0, 1, 2, 3, 4, 5, 6 and 7g/kg of mulberry leaf diethyl ether extract feed, respectively. After 30 days, 30 fish were randomly selected from each treatment group, and equally distributed to 3 fish tanks (10 fish per tank); copper sulfate is added into each fish tank water body to ensure that the concentration of the copper sulfate is 0.7mg Cu/L. Feeding fish meal soybean meal type omnivorous fish feed, and examining the feeding rate of each treatment group after four days and performing feeding recovery broken line regression analysis, the results of which are shown in Table 4 and FIG. 13. Table 4 data are expressed as mean ± standard deviation of 3 replicates; the different superscript letters in the same column data indicate significant differences, the same indicating insignificant differences (P < 0.05). The data in figure 13 are presented as an average of 3 replicates. Feeding rate (%) = feeding amount/body weight×100, feeding recovery rate (%) = (E-Y)/(K-Y) ×100.

TABLE 4 Effect of feeding Mulberry leaf Ether extract (EEE) on feeding rate of Carassius auratus after copper sulfate stress

As can be seen from Table 4, feeding the feed containing the mulberry leaf diethyl ether extract restored the feeding rate of the copper sulfate-induced crucian carp. As shown in FIG. 13, the optimal additive concentration of the mulberry leaf diethyl ether extract for recovering the feeding rate of crucian is 4.52g/kg of feed.

Example 12

Under the same cultivation environment as in example 5, 480 fish fries with a weight of 10.5+/-0.3 g are selected and randomly divided into 8 treatment groups, wherein each group comprises 4 fish tanks and 15 fish fries per tank. 8 treatment groups were fed with 0, 1, 2, 3, 4, 5, 6 and 7g/kg of mulberry leaf diethyl ether extract feed, respectively. After 30 days, 30 fish were randomly selected from each treatment group, and the fish were equally distributed to 3 fish tanks (10 fish per tank), and trichlorfon was added to the water in each fish tank to a concentration of 2.2mg/L. Four days later, the number of the fish on the side of each treatment group was examined and a fold line regression analysis of the side turn inhibition rate was performed, and the results are shown in Table 5 and FIG. 14. The data in table 5 are expressed as the mean ± standard deviation of 3 replicates, each replicate being the mean of 4-day rollover rates; the different superscript letters in the same column data indicate significant differences, the same indicating insignificant differences (P < 0.05). The data in figure 14 are presented as an average of 3 replicates. Rollover rate (%) =number of rollover fish/total number of fish×100.

TABLE 5 Effect of feeding Mulberry leaf Ether extract (EEE) on the Carassius auratus rollover Rate after dipterex stress

As can be seen from Table 5, feeding the mulberry leaf diethyl ether extract inhibited trichlorfon-induced rollover of the crucian. As shown in FIG. 14, the optimal additive concentration of the mulberry leaf diethyl ether extract for inhibiting the rollover of crucian is 4.90g/kg of feed.

Although specific embodiments of the invention have been described in detail with reference to the accompanying drawings, it should not be construed as limiting the scope of protection of the present patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.

Claims (7)

1. Application of folium Mori extract in preparing product for inhibiting freshwater fish culture stress is provided; the mulberry leaf extract is added into feed for use, and the adding proportion is 4.52-7.00 wt%o; the mulberry leaf extract is mulberry leaf diethyl ether extract; the stress is high-density cultivation resistance, copper sulfate and trichlorfon stress;

the mulberry leaf diethyl ether extract is prepared by the following method: mixing the first filter residue obtained in the extraction process of Sang Shehuan hexane extract with diethyl ether according to a ratio of 1: mixing 5-8, stirring, extracting, filtering, repeatedly extracting for 2-3 times to obtain residue-free filtrate and second filter residue, mixing the filtrates, and distilling under reduced pressure to constant weight to obtain the product;

the Sang Shehuan hexane extract is prepared by the following method: drying and crushing mulberry leaves, and mixing mulberry leaf powder and cyclohexane according to a ratio of 1: mixing 5-8, stirring, extracting, filtering, repeatedly extracting for 2-3 times to obtain filtrate and first filter residue, mixing the filtrates, and distilling under reduced pressure to constant weight.

2. An anti-stress agent for freshwater fish, comprising the mulberry leaf extract of claim 1.

3. A feed comprising a basal feed and the mulberry leaf extract of claim 1.

4. A feed as claimed in claim 3, comprising the following components in parts by weight: the mulberry leaf extract accounts for 4.52-7.00 per mill of the total weight of the feed.

5. The feed as claimed in claim 4, which comprises the following components in parts by weight: the mulberry leaf extract accounts for 4.90 per mill of the total weight of the feed.

6. The feed of any one of claims 3-5, wherein the basal feed comprises the following components in parts by weight: 23.00-25.00 parts of fish meal, 31.00-33.00 parts of bean pulp, 0.60-0.80 part of DL-methionine, 36.00-38.00 parts of flour, 3.00-3.50 parts of grease, 0.80-1.30 parts of vitamin additive and 0.80-1.30 parts of mineral additive.

7. The method for preparing the feed according to any one of claims 3 to 6, comprising the steps of:

dissolving folium Mori extract in oil, mixing with other components of basic feed, and granulating.

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