阅读说明：本技术 一种促进蚂蟥生长的金属元素添加方法 (Method for adding metal elements for promoting leech growth ) 是由 郭巧生 史红专 施国伟 于 2018-06-15 设计创作，主要内容包括：本发明提供一种促进蚂蟥生长的金属元素添加方法。本发明目的在现有室内蚂蟥养殖技术基础上提供一种能显著提高蚂蟥体质量,增强免疫力,增加经济效益的养殖方法。采用本发明方法养殖蚂蟥,可比现有养殖蚂蟥体质量提高约22.07％,死亡率降低37.5％。与现有蚂蟥养殖相比,不完全统计,采用本发明方法养殖蚂蟥每亩可增加蚂蟥干重约31.55公斤,按照目前市场价1000元/kg计,每亩增加经济效益约3.16万元。(The invention provides a method for adding a metal element for promoting leech growth. The invention aims to provide a leech culture method which can obviously improve the quality of leeches, enhance the immunity and increase the economic benefit on the basis of the existing indoor leech culture technology. Compared with the prior leech culture method, the quality of the leech culture method is improved by about 22.07%, and the death rate is reduced by 37.5%. Compared with the existing leech culture, the leech culture method has the advantages that the leech culture method can increase the leech dry weight by about 31.55 kilograms per mu, and the economic benefit is increased by about 3.16 ten thousand yuan per mu according to the current market price of 1000 yuan per kg.)

1. A method for adding metal elements for promoting leech growth is characterized by comprising the following steps: in the normal leech breeding process, a certain concentration of metal elements are added to promote the growth of leeches, enhance the immunity and reduce the death rate.

2. The method for adding the metallic element for promoting the growth of leeches, according to claim 1, wherein the metallic element comprises the following components in percentage by weight: leeches cultivated are Whitmania pigra Whitman.

3. The method for adding the metallic element for promoting the growth of leeches, according to claim 1, wherein the metallic element comprises the following components in percentage by weight: the trace element is metal zinc ion.

4. The method for adding the metallic element for promoting the growth of leeches, according to claim 1, wherein the metallic element comprises the following components in percentage by weight: the concentration of zinc ions in the aquaculture water body is 0.5 mg.L^-1。

5. The method for adding the metallic element for promoting the growth of leeches, according to claim 1, wherein the metallic element comprises the following components in percentage by weight: keeping the concentration of zinc ions in the aquaculture water body to be 0.5 mg.L^-1The addition of zinc ions was stopped after 50 days.

6. The method for adding the metallic element for promoting the growth of leeches, according to claim 1, wherein the metallic element comprises the following components in percentage by weight: leeches are harvested at least after the zinc ion addition is stopped and normal culture is carried out for 40 days.

One, the technical field

The invention relates to a method for adding a metal element for promoting leech growth, and belongs to the field of medicinal animal breeding.

Second, background Art

Leech (Whitmania Pigra Whitman) is one of the basic animals of medicinal leech in Chinese pharmacopoeia (2015 edition), has the effects of breaking blood and eliminating symptoms, removing blood stasis and stimulating the menstrual flow after being dried, and is a main raw material of a plurality of Chinese patent medicines for treating cardiovascular diseases. With the continuous and deep research on leech breeding technology, the indoor artificial breeding method has achieved primary effect.

Researches find that the natural leech bait, namely pearicaspia (Bellamya puliformis) has higher enrichment effect on heavy metal ions, and Zn in body fluid of the leech natural bait, namely the pearicaspia (Bellamya puliformis) is²⁺The content of the active ingredients reaches 12.12 mg/kg^-1. Zinc, as an essential element of organisms, plays an important role in stabilizing a multilevel structure and in the processes of replication and transcription of genetic materials, and has important significance in maintaining the structure and the function of cell membranes in internal environment. But can cause body toxicity when concentration concentrations build up above ecological thresholds. At present, the indoor artificial leech breeding work is successful initially, but Zn is rarely seen²⁺Report on promoting leech growth. Determine to be favorable for leech growthZn of (2)²⁺The water bath mode can promote the increase of the quality of the leech body, and improves the indoor breeding efficiency of the leech.

Third, the invention

The invention aims to provide a leech culture method which is based on the existing indoor leech culture method, can obviously improve the quality of leech bodies, enhance the immunity and increase the economic benefit.

According to the invention, zinc ions are added into the leech culture water body to ensure that the culture water body Zn²⁺Is 0.5 mg.L^-1Culturing for 50 days continuously, and then stopping adding zinc ions, wherein other culture modes are unchanged. During the period, the room temperature is kept at 25 +/-1 ℃, 1 time of water change is carried out in 3 days, 24 hours of aerated tap water is used, and the water change time is 9 a.m.: about 00 percent of water is changed every time, and residual snails are removed. The leeches which die are found in the breeding process are timely cleaned.

Through experimental statistics and comparison, the Zn is added by adopting the method²⁺Compared with the existing leech culture method, the leech culture method has the advantages that the quality of the leech culture method is improved by about 22.07%, and the death rate is reduced by 37.5%. Incomplete statistics shows that the leech dry weight can be increased by about 31.55 kilograms per mu, and the economic benefit can be increased by about 3.16 ten thousand yuan per mu according to the current market price of 1000 yuan/kg.

Description of the drawings

FIG. 1: zn²⁺The effect of enrichment and release on the specific growth rate of leeches.

FIG. 2: zn²⁺Influence of enrichment and release on leech digestive enzyme activity Zn²⁺The enrichment and release influence on the leech digestive enzyme activity (A: lipase, B: amylase, C: acid protease, D: alkaline protease).

FIG. 3: zn²⁺Enrichment and release effects on the anti-lipase activity of leeches (A: superoxide dismutase, B: catalase, C: alkaline phosphatase).

Different lower case letters indicate significant differences (P < 0.05) between different groups at the same time period, and different upper case letters indicate significant differences (P < 0.05) between different time periods in the same group.

Fifth, detailed description of the invention

1. Material

1.1 Experimental materials and instruments:

leeches are provided by the institute of traditional Chinese medicine of Nanjing agriculture university and identified as leeches W.

Alpha-1506 ultraviolet visible spectrophotometer (Shanghai spectral light instruments Co., Ltd.), 5810R centrifuge (Eppendorf Co., Ltd.), XHF-D homogenizer (Ningbo New Ganoderma Biotech Co., Ltd.), FYL-YS-108L cryopreservation box (Beijing Foley electric appliances Co., Ltd.), ZD-2 type automatic potentiometric titrator (Shanghai instruments electro scientific instruments Co., Ltd.), FA 1104 analytical balance (Shanghai precision scientific instruments Co., Ltd.), KQ250B ultrasonic instrument (Suzhou Kunshan ultrasonic instruments Co., Ltd.), GXZ intelligent light incubator (Ningbo Jiangnan instruments factory), inductively coupled plasma emission spectrometer (2100DC, USA), and the like.

2 method

2.1 design of leech growth experiment

Uniformly selecting about 4g of healthy leeches in Zn²⁺On the basis of acute toxicity test of leeches, a water body Zn is arranged²⁺The concentrations were 0.3, 0.4, 0.5, 0.6 and 0.7 mg.L^-1Five Zn²⁺Mass concentration group and a blank control group, each group has 30 bottles, and each bottle has 10 strips. The other daily breeding and management conditions were the same, the experimental time was 50 days, and the body weight was measured at the initial and final stages. Screening and determining Zn most suitable for leech growth²⁺And (4) concentration.

On the basis of the test, 3-4g of robust leeches are uniformly selected, and Zn with proper concentration is added into the water body²⁺Is Zn²⁺Groups, while normal breeding was control group, each group had 30 bottles with 10 strips per bottle. The two groups of other daily breeding and management conditions are the same, the experimental time is 100 days, wherein certain concentration Zn is added in the first 50 days²⁺Is Zn²⁺In enrichment period, stopping adding Zn after 50 days²⁺A release period. Measuring body weight and Zn of different tissues every 10 days²⁺Content and digestive tract digestive enzyme activity, and the inherent quality of leeches is detected on the 50 th day (end of enrichment period) and the 100 th day (end of release period).

In a 100-day culture experiment, the Specific Growth Rate (SGR) and Survival Rate (SR) of leeches every 10 days are calculated according to the following formulas. Wherein Wt is the average mass (g) on day T of the experiment, day T (10d) of the experiment, and W (T +10) is the average mass (g) on day T +10 of the experiment.

SR (%) < 100 × (total number-number of dead pieces)/total number of pieces

SGR(％)＝100×(lnW(t+10)-lnWt)/T

2.2 determination of digestive enzyme Activity

Cleaning with pre-cooled distilled water, sucking water from surface of digestive tract tissue, homogenizing in ice bath, adding distilled water, diluting to obtain 20% solution, centrifuging at 4 deg.C for 15min (rotation speed of 8000r min)^-1) And the obtained supernatant is enzyme solution.

The amylase activity is measured by a 3, 5-dinitrosalicylic acid colorimetric method, and the yield of 1mg of reducing sugar generated by hydrolyzing starch at 1min of enzyme amount per unit volume at 37 ℃ is 1 amylase activity unit; the lipase activity adopts a p-nitrophenylpalmitate (rho-NPP) colorimetric method, and the enzyme quantity for catalytically releasing 1 mu mol of p-nitrophenol in 1min at 37 ℃ is defined as 1 enzyme activity unit; protease activity Using the Folin-phenol method, it is specified that hydrolysis of casein per minute produces 1 μ g of tyrosine, defined as 1 protease activity unit. The protein content of the tissue homogenate was measured by a coomassie brilliant blue G-250 colorimetric method.

Defining the number of units of enzyme activity per mg of soluble enzyme protein (U.mg)^-1) Is 1 enzyme specific activity (U.mg)^-1·prot^-1)。

2.3 different textures of leech Zn²⁺Determination of the content

Taking Zn in 2.1 enrichment and release experiments every 10 days²⁺And performing microwave digestion on the skin, muscle and digestive tract of leeches in a control group, and then determining Zn by adopting an inductively coupled plasma mass spectrometry method²⁺And (4) content.

2.4 intrinsic quality determination

Collecting leeches at the final enrichment and release stages, and measuring the water content, the total ash content, the acid-insoluble ash content, the pH value and the antithrombin activity of the leeches according to a method of 'Chinese pharmacopoeia' of 2015 edition.

2.5 statistical methods

The data from the experiment were initially processed using Microsoft Excel and then analyzed using the SPSS 20.0 software package (One-Way ANOVE, LSD).

3 results and analysis

3.1 different concentrations of Zn²⁺Influence on leech growth

Different concentrations of Zn²⁺The effect on leech growth is shown in table 1. As can be seen from Table 1: zn within 20-30 days in the early period of the experiment²⁺Group removal 0.5-0.7 mg.L on day 20^-1The other concentrations of Zn outside the concentration group²⁺The specific growth rate of the leech group is obviously higher than that of the control group (P < 0.05). Zn at 40 days²⁺The concentration is higher than 0.5 mg.L^-1The specific growth rate of leeches in the group is lower than that of the control group, and the specific growth rate of the ion group is not higher than that of the control group at the 50 th day.

TABLE 1 different concentrations of Zn²⁺Influence on weight gain of leeches

Note: different lower case letters indicate that the different groups differed significantly (P < 0.05) at the same time period. Table 2 is the same as table 4.

3.2 Zn²⁺Effect of enrichment and release on leech growth

On the basis of 3.1, 0.5 mg-L leech with the best influence on the growth of leeches is screened^-1Zn²⁺Group, continue to observe Zn²⁺The influence of enrichment and release on the growth performance of leeches. Zn²⁺The effect of enrichment and release on leech body weight and mortality is shown in table 2 and the effect on specific growth rate is shown in figure 1.

As can be seen from Table 2, Zn was released from the 10 th day to the 100 th day of the enrichment at the final stage²⁺The weight of the leech in the group is obviously larger than that of the leech in the control group (P is less than 0.05). Zn²⁺The death rate of leeches in the enrichment and release processes is far less than that of a control group; the mortality rate during enrichment is less than during release.

TABLE 2 Zn²⁺Effect of enrichment and Release on leech body weight and mortality

As can be seen from FIG. 1, Zn is present as the enrichment time is prolonged²⁺The difference between the specific growth rate of the group and the contrast group is gradually reduced, and Zn is enriched within 40 days²⁺The specific growth rate of the leeches in the group is obviously higher than that of the control group (P is less than 0.05), and the specific growth rate of each group is not obviously different on the 50 th day of enrichment (P is more than 0.05). Ion release period Zn²⁺No significant difference (P > 0.05) exists between the specific growth rate of the group and the control group.

Note: different lower case letters indicate significant differences (P < 0.05) between different groups at the same time period, and different upper case letters indicate significant differences (P < 0.05) between different time periods in the same group. FIGS. 2-3 and Table 3 are the same. 3.3 Zn²⁺Influence of enrichment and release on leech digestive enzyme

Zn²⁺The effect of enrichment and release on leech digestive enzyme activity is shown in figure 2. As can be seen from FIG. 2, control group and Zn²⁺The activity of the group digestive enzyme is increased firstly and then decreased, and reaches the maximum value in 30-50 days. Zn in the first 40 days²⁺The leech digestive enzyme is obviously larger than a control group (P is less than 0.05) except lipase, and Zn²⁺Alkaline protease is obviously higher than that of a control group (P is less than 0.05) at the 50 th day of the group, and Zn is remained in the rest time period²⁺All digestive enzyme activities in the group have no significant difference (P > 0.05) with the control group.

3.4 Zn²⁺Influence of enrichment and release on leech adverse resistance enzyme

Zn²⁺The effect of the enrichment and release on the anti-retroenzyme of leeches is shown in figure 3. As can be seen from FIG. 3, Zn²⁺The influence on the leech adverse enzyme activity is as follows: zn²⁺The SOD and AKP activities of the group are obviously higher than those of the control group (P is less than 0.05) within 30 days from the beginning of enrichment, and Zn is added in the rest time period²⁺The group has no significant difference (P is more than 0.05) from the control group; zn²⁺CAT activity of the group is obviously higher than that of the control group (P is less than 0.05) within 40 days from the beginning of enrichment, and Zn is added in the rest time period²⁺The group and the control group have no significant difference (P > 0.05).

3.5 different textures of leech Zn²⁺Enrichment and Release Pattern

Different tissue pairs of leeches Zn²⁺The enrichment and release profiles are shown in Table 3. As can be seen from Table 3, the leeches of the control group have different structures Zn^{2 +}The content of the extract increases along with the time, and gradually stabilizes until about 90 days; zn²⁺Group different tissue Zn²⁺The content enrichment period and the release period are both significantly different (P is less than 0.05) in 20 days, wherein the enrichment period is increased, the release period is decreased, and the content gradually stabilizes around 30 days of the release period. The whole enrichment period and the first 30 days of the release period, and the same Zn period²⁺The content of different tissues in the groups is obviously higher than that of the control group (P is less than 0.05), and two groups of Zn begin to release at the 40 th day²⁺The contents all gradually become stable and have no significant difference (P is more than 0.05).

As can be seen from Table 3, Zn²⁺The content in different tissues is skin < muscle < digestive tract, wherein the content in the digestive tract and the muscle is obviously higher than that in the skin (P < 0.05), and the content in the digestive tract and the muscle has no obvious difference (P > 0.05). Zn compared with the control group²⁺Zn in the skin, muscle and digestive tract of the group²⁺The increase of the amount of the final stage of enrichment was 43.50 mg/kg^-1、59.87 mg·kg^-1And 57.00 mg/kg^-1At the final stage of release-60.60 mg/kg^-1、-58.05mg·kg^-1And-58.25 mg kg^-1It can be known that leeches have different tissue pairs Zn²⁺The enrichment capacity is muscle > digestive tract > skin, and the release capacity is skin > digestive tract > muscle.

TABLE 3 Zn²⁺Enriching and releasing Zn in different tissues of leeches²⁺Effect of content (mg. kg)^-1)

3.6 Zn²⁺Influence of enrichment and release on inherent quality of leeches

Zn²⁺The effect of enrichment and release on the intrinsic quality of leeches is shown in table 4. As can be seen from Table 4, Zn²⁺The intrinsic qualities of acid-insoluble ash, antithrombin activity and the like at the end of enrichment and the end of release of the group are not obviously different from those of a control group (P is more than 0.05).

TABLE 4 Zn²⁺Influence of enrichment and release on inherent quality of leeches

4 comprehensive judgment

The experimental result shows that the adding concentration is 0.5 mg.L^-1Zn²⁺The leech culture medium can improve the growth performance and the immunocompetence of the leech, can improve the physique of the leech by about 22.07% compared with the existing leech culture medium, reduces the death rate by 37.5%, and has no obvious influence on the inherent quality of the leech. However, since leeches are medicinal animals, ions need to be released for at least 40 days to achieve the safety as much as possible. Compared with the prior leech culture, the leech culture method has the advantages that 0.5mg L is added into the leech culture water body^-1Zn²⁺The leech dry weight can be increased by about 31.55 kilograms per mu in 50 days, and the economic benefit is increased by about 3.16 ten thousand yuan per mu according to the current market price of 1000 yuan/kg.