阅读说明：本技术 基于人工养殖中国鲎的可持续采血方法 (Sustainable blood sampling method based on artificial culture of Tachypleus tridentatus ) 是由 贾久满 廖永岩 李伟峰 李健 张艳秋 方怀义 吴卓林 于 2020-05-25 设计创作，主要内容包括：一种基于人工养殖中国鲎的可持续采血方法,包括养殖管理-采血监管；养殖场地包括人工养殖池和治疗恢复池；人工养殖池修建于海域附近,按人工养殖池面积的10%在人工养殖池的附近配备治疗恢复池；每次进行采血作业以后,指标低的中国鲎放入治疗恢复池,暂停采血作业,恢复后可以继续采血并放入人工养殖池中。这种采血方法基于人工养殖的中国鲎,改变传统的野生采血方式,采用人工养殖条件下的中国鲎,通过养殖管理和采血监管为一体的管理体系,实现可持续采血,能够有效保护中国鲎资源、确保鲎试剂产业健康发展；人工养殖条件下可持续采血,即定期对鲎进行采血,确保鲎的成活率,提高鲎的利用率,实现可持续多次采血,减少中国鲎的捕获量。(A sustainable blood sampling method based on artificial cultivation of Tachypleus tridentatus comprises cultivation management-blood sampling supervision; the culture site comprises an artificial culture pond and a treatment recovery pond; the artificial culture pond is built near the sea area, and a treatment recovery pond is arranged near the artificial culture pond according to 10% of the area of the artificial culture pond; after each blood sampling operation, the Tachypleus tridentatus with low index is put into a treatment recovery pool, the blood sampling operation is suspended, and the blood can be continuously sampled after recovery and put into an artificial culture pool. The blood sampling method is based on artificially cultured Tachypleus tridentatus, changes the traditional wild blood sampling mode, adopts Tachypleus tridentatus under artificial culture conditions, realizes sustainable blood sampling through a management system integrating culture management and blood sampling supervision, and can effectively protect Tachypleus tridentatus resources and ensure the healthy development of Tachypleus tridentatus reagent industry; the blood can be continuously collected under the artificial culture condition, namely, the blood is collected from the horseshoe crab periodically, the survival rate of the horseshoe crab is ensured, the utilization rate of the horseshoe crab is improved, the blood can be continuously collected for many times, and the capture amount of the horseshoe crab in China is reduced.)

1. A sustainable blood sampling method based on artificial culture of Tachypleus tridentatus is characterized by comprising the following steps: the sustainable blood sampling method comprises cultivation management-blood sampling supervision;

the cultivation management comprises cultivation site design and cultivation environment management

The culture site comprises an artificial culture pond and a treatment recovery pond; the artificial culture pond is built near the sea area, and a treatment recovery pond is arranged near the artificial culture pond according to 10% of the area of the artificial culture pond;

the cultivation environment management comprises the following steps:

(1) culturing density: not more than 1/m²；

(2) Salinity: 15-30 per mill;

(3) the dissolved oxygen is above 4 mg/L;

(4) feeding: feeding for 1 time every day, wherein the feeding amount is 2-3% of the weight of the tachypleus tridentatus;

(5) feed: adopting iced fresh baits or artificial compound feed of marine trash fish, shellfish and the like;

the blood sampling supervision comprises preparation before blood sampling, health examination and supervision after blood sampling:

preparation before blood sampling

Heating the blood taking needle and the blood taking bottle at 250 ℃ for more than 30 min to remove pyrogen, and putting an anticoagulant with the same volume as the blood to be taken into the blood taking bottle for later use;

health examination

Judging the health condition of the tachypleus tridentatus according to the activity state of the tachypleus tridentatus, and checking the healing state of the pinhole at the cardiac valve;

post-blood collection supervision

After each blood sampling operation, a small amount of blood sample is taken to detect the indexes such as the number of blood cells, and the number of blood cells is less than 20 × 10³Placing Tachypleus tridentatus (L.) Druce of one/ml into the treatment recovery pool, and suspending blood collection operation until the number of blood cells is recovered to 30 × 10³More than one per ml, blood can be continuously collected and put into an artificial culture pond.

2. The sustainable blood collection method based on artificial culture of horseshoe crab of claim 1, wherein: the artificial culture pond is built near a sea area with good water quality, sea sand with the particle size of 0.2-2.5 mm and the thickness of 8-10 cm is paved on the hard bottom of cement and the like in the pond, and the water depth in the pond is 1.5-2.0 m.

3. The sustainable blood collection method based on artificial culture of horseshoe crab of claim 1, wherein: the treatment and recovery pond is a cement pond, sea sand with particle size of 0.2-2.5 mm, thickness of 8-10 cm and area less than 30m is laid on the bottom²The water depth is 1-1.2 m.

4. The sustainable blood collection method based on artificial culture of horseshoe crab of claim 1, wherein: the blood sampling process is as follows,

catching the Tachypleus tridentatus from the artificial culture pond, cleaning the Tachypleus tridentatus by using clean seawater, and then putting the Tachypleus tridentatus on a workbench frame of a sterile room for fixing;

disinfecting the cardiac position at the chest and abdomen joint of the back side of the patient by using an iodophor disinfectant, and wiping the iodophor by using 75% alcohol;

puncturing the heart valve of a Chinese horseshoe crab with a blood taking needle to allow the blood of the horseshoe crab to flow into a blood taking bottle;

after the blood sampling is finished, the needle head is pulled out, the needle hole is sealed by the adhesive bandage, and the needle is placed back into the pond.

5. The sustainable blood collection method based on artificial culture of horseshoe crab of claim 4, wherein: blood is collected once every a period of time, and the blood collection interval time is not less than 2 months.

6. The sustainable blood collection method based on artificial culture of horseshoe crab of claim 4, wherein: the maximum blood sampling amount is completed according to the following indexes according to the blood sampling period,

the blood sampling amount of female limulus is 40 m L/kg and the blood sampling amount of male limulus is 50 m L/kg in 3-11 months, and the blood sampling amount of female limulus is 30m L/kg and the blood sampling amount of male limulus is 40 m L/kg in 12-2 months next year.

7. The sustainable blood collection method based on artificial culture of horseshoe crab of claim 1, wherein: the anticoagulant is a mixed solution of theophylline and NaCl solution.

8. The sustainable blood collection method for horseshoe crab cultivation by the artificial breeding of claim 7, wherein the salt content of the anticoagulant is 25 per mill and the theophylline concentration is 0.5 mol/L, wherein the anticoagulant is prepared by mixing theophylline (concentration is more than or equal to 98%) and NaCl solution.

Technical Field

The invention relates to a blood sampling method of Tachypleus tridentatus, in particular to a sustainable blood sampling method based on artificial culture of Tachypleus tridentatus.

Background

The northern bay is the most important habitat of Tachypleus tridentatus and is also the only area with the largest number of Tachypleus tridentatus stored in the world, only Tachypleus tridentatus and Limulus polyphemus can produce the Limulus tridentatus reagent in the world, so the Limulus tridentatus reagent can be divided into two according to the source, one is the Tachypleus tridentatus reagent (TA L) and the raw material is the blood of Tachypleus tridentatus and the other is the Limulus tridentatus reagent (L imus tridentatus L ysate and L A L) and the raw material is the blood of the Limulus tridentatus, the Limulus tridentatus is the largest and most suitable for producing the Limulus tridentatus reagent in the world, the Limulus tridentatus can reach 60 cm in length, the body weight of 3 kg-5 kg is composed of three parts, the Thymus tridentatus (so called Limulus tridentatus) and the tail is the world, the Limulus tridentatus gigas, the Limulus tridentatus gigas, the.

Since Tachypleus tridentatus is an important marine medicinal biological resource for producing a limulus reagent, the Tachypleus tridentatus resource in the northern gulf sea area is almost exhausted, and the protection of the Tachypleus tridentatus is not only required for species protection, but also required for the healthy development of industrial economy. The limulus reagent is widely applied to endotoxin detection of medicines, sanitation and foods, the demand is continuously increased, and the protection of the Tachypleus tridentatus poses a greater challenge.

Because of the rigid demand of China on limulus reagents, the limulus tridentatus serves as an important strategic biological resource of China, and the population of the limulus tridentatus needs to be rescued, restored and protected urgently. For example, adult horseshoe crabs require 5000 blood sources per year, which are produced by horseshoe crab reagent companies in an annual amount of 150 ten thousand. Under conservative estimation, the requirement of China on limulus reagents reaches more than 1000 million each year, at least 3 million adult China limulus needs to ensure the normal supply of the limulus reagents each year, and currently, no limulus reagent substitute which can be popularized and used in the market exists. However, the number of Tachypleus tridentatus as a raw material is drastically reduced, and the demand of the market for Tachypleus tridentatus blood is far from being satisfied.

At present, a conventional mode for collecting the limulus blood mainly comprises that people capture the limulus when the limulus approaches the coast while the limulus is handed over, the blood volume of 1/5-1/3 is collected, about 100ml of blood is collected from each limulus, and the limulus who has collected the blood is released to a place which is about 120km away from the captured place on the same day, so that the limulus is prevented from being captured again in a short time.

Disclosure of Invention

The invention provides a method for realizing sustainable blood sampling of Tachypleus tridentatus based on artificial culture conditions, aiming at various defects of a traditional method for taking blood of Tachypleus tridentatus in the background art.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a sustainable blood sampling method based on artificial culture of Tachypleus tridentatus comprises culture management-blood sampling supervision;

the cultivation management comprises cultivation site design and cultivation environment management

The culture site comprises an artificial culture pond and a treatment recovery pond; the artificial culture pond is built near the sea area, and a treatment recovery pond is arranged near the artificial culture pond according to 10% of the area of the artificial culture pond;

the cultivation environment management comprises the following steps:

(1) culturing density: not more than 1/m²；

(2) Salinity: 15-30 per mill;

(3) the dissolved oxygen is above 4 mg/L;

(4) feeding: feeding for 1 time every day, wherein the feeding amount is 2-3% of the weight of the tachypleus tridentatus;

(5) feed: adopting iced fresh baits or artificial compound feed of marine trash fish, shellfish and the like;

the blood sampling supervision comprises preparation before blood sampling, health examination and supervision after blood sampling:

preparation before blood sampling

Heating the blood taking needle and the blood taking bottle at 250 ℃ for more than 30 min to remove pyrogen, and putting an anticoagulant with the same volume as the blood to be taken into the blood taking bottle for later use;

health examination

Judging the health condition of the tachypleus tridentatus according to the activity state of the tachypleus tridentatus, and checking the healing state of the pinhole at the cardiac valve;

post-blood collection supervision

After each blood sampling operation, a small amount of blood sample is taken to detect the indexes such as the number of blood cells, and the number of blood cells is less than 20 × 10³Placing Tachypleus tridentatus (L.) Druce of one/ml into the treatment recovery pool, and suspending blood collection operation until the number of blood cells is recovered to 30 × 10³More than one per ml, blood can be continuously collected and put into an artificial culture pond.

The artificial culture pond is built near a sea area with good water quality, sea sand with the particle size of 0.2-2.5 mm and the thickness of 8-10 cm is paved on the hard bottom of cement and the like in the pond, and the water depth in the pond is 1.5-2.0 m.

The treatment and recovery pond is a cement pond, sea sand with particle size of 0.2-2.5 mm, thickness of 8-10 cm and area less than 30m is laid on the bottom²The water depth is 1-1.2 m.

The blood sampling process is as follows,

catching the Tachypleus tridentatus from the artificial culture pond, cleaning the Tachypleus tridentatus by using clean seawater, and then putting the Tachypleus tridentatus on a workbench frame of a sterile room for fixing;

disinfecting the cardiac position at the chest and abdomen joint of the back side of the patient by using an iodophor disinfectant, and wiping the iodophor by using 75% alcohol;

puncturing the heart valve of a Chinese horseshoe crab with a blood taking needle to allow the blood of the horseshoe crab to flow into a blood taking bottle;

after the blood sampling is finished, the needle head is pulled out, the needle hole is sealed by the adhesive bandage, and the needle is placed back into the pond.

Blood is collected once every a period of time, and the blood collection interval time is not less than 2 months.

The maximum blood sampling amount is completed according to the following indexes according to the blood sampling period,

the blood sampling amount of female limulus is 40 m L/kg and the blood sampling amount of male limulus is 50 m L/kg in 3-11 months, and the blood sampling amount of female limulus is 30m L/kg and the blood sampling amount of male limulus is 40 m L/kg in 12-2 months next year.

The anticoagulant is a mixed solution of theophylline and NaCl solution, the theophylline (the concentration is more than or equal to 98%) and the NaCl solution are mixed to prepare the anticoagulant, the salinity of the anticoagulant is 25 per mill, and the theophylline concentration is 0.5 mol/L.

Compared with the prior art, the blood sampling method disclosed by the invention is based on artificially cultured Tachypleus tridentatus, changes the traditional wild blood sampling mode, adopts Tachypleus tridentatus under the artificial culture condition, realizes sustainable blood sampling through a management system integrating culture management and blood sampling supervision, and can effectively protect Tachypleus tridentatus resources and ensure the healthy development of Tachypleus tridentatus reagent industry; the blood can be continuously collected under the artificial culture condition, namely, the blood is collected from the horseshoe crab periodically, the survival rate of the horseshoe crab is ensured, the utilization rate of the horseshoe crab is improved, the blood can be continuously collected for many times, and the capture amount of the horseshoe crab in China is reduced.

Detailed Description

The present invention will be further described with reference to the following examples.

The invention aims at implementing sustainable blood sampling for artificially cultured Tachypleus tridentatus, and comprises culture management and blood sampling supervision.

The cultivation management comprises cultivation site design and cultivation environment management; the blood sampling supervision comprises preparation before blood sampling, health examination and supervision after blood sampling.

1. Design of breeding field

1.1 Artificial culture pond

Sea sand with particle size of 0.2-2.5 mm, thickness of 8-10 cm and water depth of 1.5-2.0 m is paved at hard bottom of pond, cement pond and the like for culturing Tachypleus tridentatus near sea area with good water quality. Healthy Tachypleus tridentatus which can be used for blood sampling operation is cultured in an artificial culture pond.

1.2 therapeutic recovery pool

Preparing a treatment recovery pond according to 10% of the area of the artificial culture pond, wherein the treatment recovery pond is a cement pond, sea sand is paved at the bottom, the particle size is 0.2-2.5 mm, the thickness is 8-10 cm, and the area is less than 30m for observing the activity of Tachypleus tridentatus²The water depth is about 1-1.2 m. Culturing Tachypleus tridentatus with unqualified health examination in a treatment recovery pond.

2. Cultivation environment and management requirements

2.1 culture density: not more than 1/m²；

2.2 salinity: 15-30 per mill;

2.3 the dissolved oxygen is more than 4 mg/L;

2.4 feeding: feeding for 1 time every day, wherein the feeding amount is 2% -3% of the body weight of Tachypleus tridentatus;

2.5 feed: adopts iced fresh baits or artificial compound feed of marine trash fish, shellfish and the like.

3. Blood sampling supervision

3.1 preparation before blood sampling

Heating the blood taking needle and the blood taking bottle at 250 ℃ for more than 30 min to remove pyrogen, and placing anticoagulant with the same volume as the blood to be taken into the blood taking bottle for later use.

The anticoagulant is a mixed solution prepared from theophylline (more than or equal to 98%) and NaCl solution, wherein the salinity of the mixed solution is 25 per mill, and the theophylline concentration of the mixed solution is 0.5 mol/L.

3.2 health examination

3.2.1 judging the health condition of the tachypleus tridentatus according to the activity state of the tachypleus tridentatus, and checking the healing state of the pinhole at the cardiac valve. Limulus tridentatus with good health and no infection after pinhole healing can be weighed and sampled according to the plan. If the health condition does not meet the blood sampling requirement, the blood is put into a treatment recovery pool after corresponding treatment is carried out until the health condition is reached.

3.2.2 after each blood sampling operation, taking a small amount of blood sample to test the indexes such as the number of blood cells, wherein the number of the blood cells is less than 20 × 10³Placing Tachypleus tridentatus (L.) Druce of one/ml into the treatment recovery pool, and suspending blood collection operation until the number of blood cells is recovered to 30 × 10³More than one per ml, blood can be continuously collected and put into an artificial culture pond.

3.3 blood sampling procedure

The method comprises the steps of fishing the Tachypleus tridentatus from a pond, cleaning the Tachypleus tridentatus with clean seawater, fixing the Tachypleus tridentatus on a workbench stand in a sterile room, disinfecting the position of a cardiac valve at the chest-abdomen joint of the back side of the Tachypleus tridentatus with iodophor disinfectant, and wiping the iodophor with 75% alcohol. Puncturing the heart valve of a Chinese horseshoe crab with a blood taking needle to allow the blood of the horseshoe crab to flow into a blood taking bottle; after the blood sampling is finished, the needle head is pulled out, the needle hole is sealed by the adhesive bandage, and the needle is placed back into the pond.

3.4 blood sampling interval time: blood is collected once every a period of time, and the interval time between adjacent blood collection is not less than 2 months.

3.5 the maximum blood sampling amount at each time is different according to different blood sampling periods, and the specific blood sampling amount data is as follows:

the blood collection amount of female horseshoe crab is 40 m L/kg and the blood collection amount of male horseshoe crab is 50 m L/kg in 3-11 months, and the blood collection amount of female horseshoe crab is 30m L/kg and the blood collection amount of male horseshoe crab is 40 m L/kg in 12-next 2 months.

The blood sampling mode disclosed by the invention changes the traditional blood sampling mode, and sustainable blood sampling under the condition of artificial breeding is the optimal method for protecting horseshoe crab resources in China and ensuring healthy development of the horseshoe crab reagent industry. The method for continuously collecting blood based on artificial culture conditions disclosed by the invention is characterized in that the Tachypleus tridentatus captured in the natural sea area is domesticated in a pond, then the Tachypleus tridentatus is periodically collected with blood, the survival rate of the Tachypleus tridentatus is ensured, the utilization rate of the Tachypleus tridentatus is improved, the blood can be continuously collected for many times, and the capture amount of the Tachypleus tridentatus is reduced. The life of a horseshoe crab can reach 60 years generally, and if sustainable blood sampling is realized, the blood sampling amount of one horseshoe crab is estimated to reach at least 20-30 times of that of a traditional blood sampling mode. Thus, under the current horseshoe crab reagent production scale, the population quantity of the horseshoe crabs can be ensured not to be reduced, and the population quantity of the horseshoe crabs can be effectively recovered even after ten years by combining the measures of breeding, releasing, artificial breeding and the like. Therefore, the sustainable blood sampling method under the artificial culture condition has important significance for protecting and utilizing Tachypleus tridentatus, and the healthy development of the Tachypleus tridentatus reagent industry is ensured while the resource of Tachypleus tridentatus is protected.