A quantitative method for the density of sea cucumber bone slices

Abstract

This invention discloses a quantitative method for sea cucumber bone fragment density, including steps such as sea cucumber weight measurement, anesthesia, ethanol dehydration, tissue dissection, bone fragment extraction, washing, and density quantification. Anesthesia allows the sea cucumber body wall to fully extend, which helps shorten the low-temperature dehydration time and obtain relatively uniform tissue blocks. Low-temperature dehydration with ethanol causes fibrosis of the sea cucumber body wall, facilitating tissue block dissection and bone fragment extraction. Different body wall tissue blocks are then cut to ensure representative sampling. A weak alkali is used to digest tissue blocks of specific sizes and masses, extracting all morphologically intact bone fragments. Finally, the number of bone fragments is quantified using an optical microscope and a counting frame, and the bone fragment density per unit mass of body wall tissue is calculated using a formula, which is then converted to the bone fragment density under the fresh weight of the sea cucumber before dehydration.

Description

Technical Field

[0001] This invention relates to an analytical method for sea cucumber bone slices, and more particularly to a quantitative method for the density of sea cucumber bone slices. Background Technology

[0002] The skeletal system of sea cucumbers (echinoderms) has evolved into tiny, dispersed ossicles, primarily composed of calcite-rich material. These ossicles serve as rigid supports for the soft tissues within the body, originating from the mesoderm. Initially forming as X-shaped structures at the body wall and tentacle bases, they subsequently develop into ossicles and calcareous rings, respectively. These ossicles are independently embedded in the dermis of the body wall and are considered remnants of ancient or embryonic skeletons. They play a crucial role in protecting blood vessels and nerve structures, as well as supporting tentacle movement. Their shape and size vary considerably among sea cucumber species, exhibiting characteristic types such as table-shaped, button-shaped, plate-shaped, C-shaped, rosette-shaped, rod-shaped, grain-shaped, and anchor-shaped structures. Therefore, they have long been an important basis for taxonomy.

[0003] sea ​​cucumber ( Apostichopus japonicus The sea cucumber (Stichopus japonicus) is my country's most representative temperate sea cucumber, exhibiting physiological and ecological behaviors such as dormancy, evisceration, regeneration, and autolysis. These behaviors are evolutionary adaptive strategies for animals to cope with adverse stresses, usually accompanied by metabolic changes and energy reorganization, and are therefore highly likely to be recorded by the skeletal system as important physiological events. Thus, the density of sea cucumber bone fragments has become an objective indicator for revealing an individual's life history, especially providing a scientific reference for age determination and verification. However, to date, there is no effective method for statistically analyzing the number of sea cucumber bone fragments, hindering further research into related scientific issues. Summary of the Invention

[0004] The present invention aims to solve the above-mentioned technical problems existing in the prior art by providing a quantitative method for the density of sea cucumber bone slices.

[0005] The technical solution of this invention is: a method for quantitatively measuring the bone density of sea cucumber, which is carried out in the following steps:

[0006] Step 1. Wipe the surface moisture of the sea cucumber dry and measure its wet weight W. i ;

[0007] Step 2. Soak the sea cucumber in a seawater solution with a mass concentration of 5-10% magnesium chloride or magnesium sulfate until the body is fully extended;

[0008] Step 3. Transfer the sea cucumber to a 70-75% (w / w) ethanol solution and refrigerate at 4°C until the dehydration rate reaches 55-70%, where D is the dehydration rate. R=100% × (W i – W d ) / W i The W d This is the weight of the sea cucumber measured after dehydration;

[0009] Step 4. Remove residual ethanol from the surface of the sea cucumber. Cut off the anterior 1 / 5 of the body, the anterior mouth end, and the posterior 1 / 5 of the body, the anal end, and then remove the internal organs to obtain the sea cucumber body wall. Then, cut along the junction of the tube feet and the tube feet of the sea cucumber body wall into the dorsal and ventral sections. Divide the dorsal and ventral sections into three smaller segments according to the anterior, middle, and posterior divisions. Finally, cut multiple equal tissue blocks with a mass of less than 0.001 g from each segment. The specific mass of each tissue block is denoted as M. n The n=1, 2, 3... represents the sequence number of the tissue block;

[0010] Step 5. Place each tissue block in a centrifuge tube, add a sodium hypochlorite solution with a mass concentration of 10-50%, and digest at 20-25°C in the dark until the tissue block turns into a white precipitate;

[0011] Step 6. After standing for 10 minutes, use a pipette to remove the supernatant, then add an equal volume of pure water and repeatedly invert and wash; repeat this step at least 5 times; to ensure that the bone fragments are intact and will not be eroded by residual weak alkaline solution, thus affecting the quantitative effect of bone fragments.

[0012] Step 7. Let stand for 10 minutes, then bring the volume of the solution in the centrifuge tube to V ml. Repeat the pipetting and repositioning of the solution 5-10 times. Immediately transfer T ml of solution onto a standard plankton counting frame, gently press the coverslip, and observe and count the number of bone fragments Pn in Fn fields of view under a 10x objective and 10x eyepiece of an optical microscope. The number of bone fragments in the centrifuge tube is calculated using formula ①.

[0013] ①

[0014] In the formula, N is the number of bone fragments contained in the centrifuge tube, and S is the area of ​​the counting frame (mm²). 2 Fs represents the microscope's field of view area, in mm. 2 Fn is the number of counting fields; V is the volume of the bone fragment sample liquid in the centrifuge tube; T is the volume of liquid in the counting frame; Pn is the number of bone fragments in the counted Fn fields;

[0015] According to the formula Calculate the bone fragment density for each tissue, fragments / g:

[0016] P=(W d × N) / (W i × M n )

[0017] The bone density of the sea cucumber before dehydration is the average bone density of n tissue blocks.

[0018] In step 3, the sea cucumber is transferred to an ethanol solution with a mass concentration of 70-75% and refrigerated at 4°C for 24 hours. Then, the sea cucumber is transferred to an ethanol solution with a mass concentration of 90-95% and refrigerated until the dehydration rate reaches 55-70%.

[0019] This invention includes steps such as sea cucumber weight determination, anesthesia, ethanol dehydration, tissue dissection, bone fragment extraction, washing, and density quantification. Anesthesia fully extends the sea cucumber body wall, which helps shorten the low-temperature dehydration time and obtain relatively homogeneous tissue blocks. Low-temperature dehydration with ethanol causes fibrosis of the sea cucumber body wall, facilitating tissue block dissection and bone fragment extraction. Different body wall tissue blocks are then cut to ensure representative sampling. Next, a weak alkali is used to digest tissue blocks of specific sizes and masses, extracting all morphologically intact bone fragments. Finally, the number of bone fragments is quantified using an optical microscope and a counting frame, and the bone fragment density per unit mass of body wall tissue is calculated using a formula, which is then converted to the bone fragment density under the fresh weight of the sea cucumber before dehydration. This method can provide a methodological reference for studying the content, distribution, and growth and development patterns of sea cucumber bone fragments at different life stages, and also has application prospects in the age identification and verification of sea cucumber age. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the dissection of dehydrated sea cucumber according to an embodiment of the present invention. Detailed Implementation

[0021] Example 1:

[0022] This invention discloses a method for quantitatively measuring the bone density of sea cucumber, which is carried out in the following steps:

[0023] Step 1. Wipe the surface moisture of the 1-year-old sea cucumber with absorbent paper, and measure its wet weight W using an analytical balance. i =20.50g;

[0024] Step 2. Soak (relax) the sea cucumber in a 5% magnesium chloride (MgCl2) seawater solution to anesthetize it until its body is fully extended;

[0025] Step 3. Transfer the sea cucumber to a 75% ethanol solution and refrigerate at 4°C for 24 hours. Then, remove the dehydrated and dead sea cucumber and blot off the surface ethanol with absorbent paper. Measure the mass W using an analytical balance. d =8.02 g, according to the dehydration rate D R =100% × (W i – W d ) / W iD R =60.88%;

[0026] Step 4. Absorb any residual ethanol from the surface of the sea cucumber, such as... Figure 1 As shown in (ad), the anterior 1 / 5 of the body, the anterior mouth end, and the posterior 1 / 5 of the body, the anal end, are cut off, and the internal organs are removed to obtain the body wall of the sea cucumber. Then, the body wall is cut into the dorsal and ventral sections along the junction of the tube feet and the parapodia. The dorsal and ventral sections are then divided into three smaller segments according to the anterior, middle, and posterior divisions. Finally, three equal tissue blocks with a mass of less than 0.001 g are cut from each segment. The specific mass of each tissue block is denoted as M. n The n=1, 2, 3……18 represents the sequence number of the tissue block;

[0027] Step 5. Place each tissue block into a 2 ml centrifuge tube, add 2 ml of 30% sodium hypochlorite (NaClO) solution, and digest in the dark at room temperature (25℃) until the tissue block turns into a white precipitate;

[0028] Preparation of the sodium hypochlorite (NaClO) solution: Rinse a 100 ml graduated cylinder with distilled water, weigh out 30 ml of NaClO solution, and transfer it along a glass rod into a 100 ml volumetric flask. Wash the graduated cylinder and glass rod three times with distilled water, and transfer the washing solution into the volumetric flask. Add distilled water to the volumetric flask to the mark, stopper the flask, and mix by inverting the flask.

[0029] Step 6. After standing for 10 minutes, use a pipette to aspirate 1.5 ml of supernatant, then add an equal volume of 1.5 ml of pure water, and repeatedly invert and wash for 1 minute; repeat this step at least 5 times.

[0030] Step 7. Let stand for 10 min, then pipette the solution to V=1 ml in the centrifuge tube. Repeatedly pipette the solution 5-10 times to ensure the bone fragments are evenly suspended in the centrifuge tube. Immediately aspirate T=0.1 ml of solution and spread it in a "Z" shape onto a standard plankton counting frame (20 mm × 20 mm). Gently press the coverslip and observe and count two pieces under a 10x objective and 10x eyepiece of an optical microscope. Count Fn=50 fields of view for each piece, and take the average value Pn. The number of bone fragments in the centrifuge tube is calculated using formula ①.

[0031] ①

[0032] In the formula, N is the number of bone fragments contained in the centrifuge tube, and S is the area of ​​the counting frame (mm²). 2 Fs represents the microscope's field of view area, in mm. 2Fn is the number of counting fields; V is the volume of the bone fragment sample liquid in the centrifuge tube; T is the volume of liquid in the counting frame; Pn is the number of bone fragments in the counted Fn fields;

[0033] According to the formula Calculate the bone fragment density for each tissue, fragments / g:

[0034] P=(W d × N) / (W i × M n )

[0035] The bone density of the sea cucumber before dehydration is the average bone density of 18 tissue blocks.

[0036] Example 2:

[0037] This invention discloses a method for quantitatively measuring the bone density of sea cucumber, which is carried out in the following steps:

[0038] Step 1. Wipe the surface moisture of the 2-year-old sea cucumber with absorbent paper, and measure its wet weight W using an analytical balance. i =50.20g;

[0039] Step 2. Soak (relax) the sea cucumber in an 8% magnesium chloride (MgCl2) seawater solution to anesthetize it until its body is fully extended;

[0040] Step 3. Transfer the sea cucumber to a 75% ethanol solution and refrigerate at 4°C for 24 hours. Then, remove the dehydrated and dead sea cucumber and blot off the surface ethanol with absorbent paper. Measure the mass W using an analytical balance. d =20.20g, according to the dehydration rate D R =100% × (W i – W d ) / W i D R =59.76%;

[0041] Step 4. Absorb any residual ethanol from the surface of the sea cucumber, such as... Figure 1 As shown in (ad), the anterior 1 / 5 of the body, the anterior mouth end, and the posterior 1 / 5 of the body, the anal end, are cut off, and the internal organs are removed to obtain the body wall of the sea cucumber. Then, the body wall is cut into the dorsal and ventral sections along the junction of the tube feet and the parapodia. The dorsal and ventral sections are then divided into three smaller segments according to the anterior, middle, and posterior divisions. Finally, three equal tissue blocks with a mass of less than 0.001 g are cut from each segment. The specific mass of each tissue block is denoted as M. n The n=1, 2, 3……18 represents the sequence number of the tissue block;

[0042] Step 5. Place each tissue block into a 2 ml centrifuge tube, add 2 ml of 30% sodium hypochlorite (NaClO) solution, and digest in the dark at room temperature (25℃) until the tissue block turns into a white precipitate;

[0043] Preparation of the sodium hypochlorite (NaClO) solution: Rinse a 100 ml graduated cylinder with distilled water, weigh out 30 ml of NaClO solution, and transfer it along a glass rod into a 100 ml volumetric flask. Wash the graduated cylinder and glass rod three times with distilled water, and transfer the washing solution into the volumetric flask. Add distilled water to the volumetric flask to the mark, stopper the flask, and mix by inverting the flask.

[0044] Step 6. After standing for 10 minutes, use a pipette to aspirate 1.5 ml of supernatant, then add an equal volume of 1.5 ml of pure water, and repeatedly invert and wash for 1 minute; repeat this step at least 5 times.

[0045] Step 7. Let stand for 10 min, then pipette the solution to V=1 ml in the centrifuge tube. Repeatedly pipette the solution 5-10 times to ensure the bone fragments are evenly suspended in the centrifuge tube. Immediately aspirate T=0.1 ml of solution and spread it in a "Z" shape onto a standard plankton counting frame (20 mm × 20 mm). Gently press the coverslip and observe and count two pieces under a 10x objective and 10x eyepiece of an optical microscope. Count Fn=50 fields of view for each piece, and take the average value Pn. The number of bone fragments in the centrifuge tube is calculated using formula ①.

[0046] ①

[0047] In the formula, N is the number of bone fragments contained in the centrifuge tube, and S is the area of ​​the counting frame (mm²). 2 Fs represents the microscope's field of view area, in mm. 2 Fn is the number of counting fields; V is the volume of the bone fragment sample liquid in the centrifuge tube; T is the volume of liquid in the counting frame; Pn is the number of bone fragments in the counted Fn fields;

[0048] According to the formula Calculate the bone fragment density for each tissue, fragments / g:

[0049] P=(W d × N) / (W i × M n )

[0050] The bone density of the sea cucumber before dehydration is the average bone density of 18 tissue blocks.

[0051] Example 3:

[0052] This invention discloses a method for quantitatively measuring the bone density of sea cucumber, which is carried out in the following steps:

[0053] Step 1. Wipe the surface moisture of the 3-year-old sea cucumber with absorbent paper, and measure its wet weight W using an analytical balance. i =90.25g;

[0054] Step 2. Soak (relax) the sea cucumber in a 10% magnesium chloride (MgCl2) seawater solution to anesthetize it until its body is fully extended;

[0055] Step 3. Transfer the sea cucumber to a 75% ethanol solution and refrigerate at 4°C for 24 hours. Remove the dehydrated and dead sea cucumber and blot the surface ethanol with absorbent paper. Measure the mass W using an analytical balance. d =62.25g, then the sea cucumber was transferred to a 95% ethanol solution and refrigerated for another 24 hours. The ethanol on the surface of the dehydrated and dead sea cucumber was then removed and the mass was measured to be W using an analytical balance. d =37.82 g, according to the dehydration rate D R =100% × (W i – W d ) / W i D R =58.09%;

[0056] Step 4. Absorb any residual ethanol from the surface of the sea cucumber, such as... Figure 1 As shown in (ad), the anterior 1 / 5 of the body, the anterior mouth end, and the posterior 1 / 5 of the body, the anal end, are cut off, and the internal organs are removed to obtain the body wall of the sea cucumber. Then, the body wall is cut into the dorsal and ventral sections along the junction of the tube feet and the parapodia. The dorsal and ventral sections are then divided into three smaller segments according to the anterior, middle, and posterior divisions. Finally, three equal tissue blocks with a mass of less than 0.001 g are cut from each segment. The specific mass of each tissue block is denoted as M. n The n=1, 2, 3……18 represents the sequence number of the tissue block;

[0057] Step 5. Place each tissue block into a 2 ml centrifuge tube, add 2 ml of 30% sodium hypochlorite (NaClO) solution, and digest in the dark at room temperature (25℃) until the tissue block turns into a white precipitate;

[0058] Preparation of the sodium hypochlorite (NaClO) solution: Rinse a 100 ml graduated cylinder with distilled water, weigh out 30 ml of NaClO solution, and transfer it along a glass rod into a 100 ml volumetric flask. Wash the graduated cylinder and glass rod three times with distilled water, and transfer the washing solution into the volumetric flask. Add distilled water to the volumetric flask to the mark, stopper the flask, and mix by inverting the flask.

[0059] Step 6. After standing for 10 minutes, use a pipette to aspirate 1.5 ml of supernatant, then add an equal volume of 1.5 ml of pure water, and repeatedly invert and wash for 1 minute; repeat this step at least 5 times.

[0060] Step 7. Let stand for 10 min, then pipette the solution to V=1 ml in the centrifuge tube. Repeatedly pipette the solution 5-10 times to ensure the bone fragments are evenly suspended in the centrifuge tube. Immediately aspirate T=0.1 ml of solution and spread it in a "Z" shape onto a standard plankton counting frame (20 mm × 20 mm). Gently press the coverslip and observe and count two pieces under a 10x objective and 10x eyepiece of an optical microscope. Count Fn=50 fields of view for each piece, and take the average value Pn. The number of bone fragments in the centrifuge tube is calculated using formula ①.

[0061] ①

[0062] In the formula, N is the number of bone fragments contained in the centrifuge tube, and S is the area of ​​the counting frame (mm²). 2 Fs represents the microscope's field of view area, in mm. 2 Fn is the number of counting fields; V is the volume of the bone fragment sample liquid in the centrifuge tube; T is the volume of liquid in the counting frame; Pn is the number of bone fragments in the counted Fn fields;

[0063] According to the formula Calculate the bone fragment density for each tissue, fragments / g:

[0064] P=(W d × N) / (W i × M n )

[0065] The bone density of the sea cucumber before dehydration is the average bone density of 18 tissue blocks.

[0066] The density of the dorsal and abdominal body wall ossicles of 1-year-old, 2-year-old, and 3-year-old sea cucumbers was determined using the methods described in Examples 1-3 above. Ten 1-year-old sea cucumbers were included, and 5 each of the 2-year-old and 3-year-old sea cucumbers were included. The results are shown in Table 1.

[0067] Table 1. Density of bone fragments in the body wall of sea cucumbers of different ages

[0068]

[0069] As shown in Table 1, the density of bone fragments on the dorsal body wall of sea cucumbers aged 1 to 3 years was higher than that on the abdominal body wall, and the bone fragment density of the body wall of 1-year-old and 2-year-old sea cucumbers was significantly higher than that of 3-year-old sea cucumbers.

[0070] Example 4:

[0071] This invention discloses a method for quantitatively measuring the bone density of sea cucumber, which is carried out in the following steps:

[0072] Step 1. Wipe less than 1 g of the surface moisture of the juvenile sea cucumber with absorbent paper, and measure the wet weight W using an analytical balance. i =0.414g;

[0073] Step 2. Soak (relax) the sea cucumber in a 5% magnesium chloride (MgCl2) seawater solution to anesthetize it until its body is fully extended;

[0074] Step 3. Transfer the sea cucumber to a 70% ethanol solution and refrigerate at 4°C for 24 hours. Remove the dehydrated and dead sea cucumber and blot the surface ethanol with absorbent paper. Measure the mass W using an analytical balance. d =0.138 g, according to the dehydration rate D R =100% × (W i – W d ) / W i D R =66.66%;

[0075] Step 4. Absorb any residual ethanol from the surface of the sea cucumber, such as... Figure 1 As shown in (ad), the anterior 1 / 5 of the body (the anterior mouth end) and the posterior 1 / 5 of the body (the anal end) are removed, and the internal organs are then removed to obtain the body wall of the sea cucumber. Next, the body wall is cut along the junction of the tube feet and the parapodia to form the dorsal and ventral sections. The dorsal and ventral sections are then divided into three smaller segments according to their anterior, middle, and posterior dimensions. Finally, three equal tissue blocks, each weighing less than 0.001 g, are cut from each segment. The dorsal tissue blocks weigh 0.0007 g, 0.0009 g, and 0.0007 g, respectively, while the ventral tissue blocks weigh 0.0006 g, 0.0007 g, and 0.0007 g, respectively. The specific weight of each tissue block is denoted as M. n The n=1,2,3……18 represents the sequence number of the tissue block;

[0076] Step 5. Place each tissue block into a 2 ml centrifuge tube, add 2 ml of 30% sodium hypochlorite (NaClO) solution, and digest in the dark at room temperature (25℃) until the tissue block turns into a white precipitate;

[0077] Preparation of the sodium hypochlorite (NaClO) solution: Rinse a 100 ml graduated cylinder with distilled water, weigh out 30 ml of NaClO solution, and transfer it along a glass rod into a 100 ml volumetric flask. Wash the graduated cylinder and glass rod three times with distilled water, and transfer the washing solution into the volumetric flask. Add distilled water to the volumetric flask to the mark, stopper the flask, and mix by inverting the flask.

[0078] Step 6. After standing for 10 minutes, use a pipette to aspirate 1.5 ml of supernatant, then add an equal volume of 1.5 ml of pure water, and repeatedly invert and wash for 1 minute; repeat this step at least 5 times.

[0079] Step 7. Let stand for 10 min, then pipette the solution to V=1 ml in the centrifuge tube. Repeatedly pipette the solution 5-10 times to ensure the bone fragments are evenly suspended in the centrifuge tube. Immediately aspirate T=0.1 ml of solution and spread it in a "Z" shape onto a standard plankton counting frame (20 mm × 20 mm). Gently press the coverslip and observe and count two pieces under a 10x objective and 10x eyepiece of an optical microscope. Count Fn=50 fields of view for each piece, and take the average value Pn. The number of bone fragments in the centrifuge tube is calculated using formula ①.

[0080] ①

[0081] In the formula, N is the number of bone fragments contained in the centrifuge tube, and S is the area of ​​the counting frame (mm²). 2 Fs represents the microscope's field of view area, in mm. 2 Fn is the number of counting fields; V is the volume of the bone fragment sample liquid in the centrifuge tube; T is the volume of liquid in the counting frame; Pn is the number of bone fragments in the counted Fn fields;

[0082] According to the formula Calculate the bone fragment density for each tissue, fragments / g:

[0083] P=(W d × N) / (W i × M n )

[0084] The bone density of the sea cucumber before dehydration is the average bone density of 18 tissue blocks.

[0085] The quantitative results of bone fragment density in each tissue block are shown in Table 2.

[0086] Table 2 Density of skeletal bone fragments from juvenile sea cucumbers with a wet weight of 0.414 g

[0087]

[0088] Table 2 shows that the body wall bone density of a juvenile sea cucumber with a wet weight of 0.414 g is 1.7088 million / g. Compared with the larger sea cucumber, the juvenile sea cucumber has a higher bone density in its body wall.

[0089] Example 5:

[0090] Using the method of Example 4, the density of characteristic bone fragments in the body wall of juvenile sea cucumber with a wet weight of 0.414g was quantified, and the results are shown in Table 3.

[0091] Table 3 Density of characteristic bone fragments in sea cucumber

[0092]

[0093] As shown in Table 3, the density of different types of bone fragments in the body wall of sea cucumber varies considerably. The density of table-shaped sea cucumbers is the highest in both the back and abdomen. The density of the same type of bone fragment also differs between the back and abdomen.

Claims

1. A method for quantitatively determining the bone density of sea cucumber, characterized in that... Follow these steps in sequence: Step 1. Wipe the surface moisture of the sea cucumber dry and measure its wet weight W. i ; Step 2. Soak the sea cucumber in a seawater solution with a mass concentration of 5-10% magnesium chloride or magnesium sulfate until the body is fully extended; Step 3. Transfer the sea cucumber to a 70-75% (w / w) ethanol solution and refrigerate at 4°C until the dehydration rate reaches 55-70%, where D is the dehydration rate. R =100% × (W i – W d ) / W i The W d This is the weight of the sea cucumber measured after dehydration; Step 4. Remove residual ethanol from the surface of the sea cucumber. Cut off the anterior 1 / 5 of the body, the anterior mouth end, and the posterior 1 / 5 of the body, the anal end, and then remove the internal organs to obtain the sea cucumber body wall. Then, cut along the junction of the tube feet and the parapodia on the sea cucumber body wall to form the back and abdomen. Divide the back and abdomen into three segments according to the anterior, middle, and posterior sections. Finally, cut multiple equal tissue blocks with a mass of less than 0.001g from each segment. The specific mass of each tissue block is denoted as M. n The n=1, 2, 3... represents the sequence number of the tissue block; Step 5. Place each tissue block in a centrifuge tube, add a sodium hypochlorite solution with a mass concentration of 10-50%, and digest at 20-25°C in the dark until the tissue block turns into a white precipitate; Step 6. After standing for 10 minutes, use a pipette to aspirate the supernatant, then add an equal volume of pure water and repeatedly invert the container to wash; repeat this step at least 5 times. Step 7. Let stand for 10 min, then bring the volume of the solution in the centrifuge tube to V ml, and then repeatedly pipet the solution 5-10 times; immediately pipet T ml of the solution onto a standard plankton counting frame, gently press the coverslip, and observe and count the number of bone fragments Pn in Fn fields under a 10x objective lens and a 10x eyepiece of an optical microscope. The number of bone fragments contained in the centrifuge tube is calculated according to formula ①: ① In the formula, N is the number of bone fragments contained in the centrifuge tube, and S is the area of ​​the counting frame (mm²). 2 Fs represents the microscope's field of view area, in mm. 2 Fn is the number of counting fields; V is the volume of the bone fragment sample liquid in the centrifuge tube; T is the volume of liquid in the counting frame; Pn is the number of bone fragments in the counted Fn fields; Calculate the bone fragment density of each tissue, in units / g, according to formula ②: P=(W d × N) / (W i × M n ) ② The bone density of the sea cucumber before dehydration is the average bone density of n tissue blocks.

2. The method for quantitatively determining the bone density of sea cucumber according to claim 1, characterized in that: In step 3, the sea cucumber is transferred to an ethanol solution with a mass concentration of 70-75% and refrigerated at 4°C for 24 hours. Then, the sea cucumber is transferred to an ethanol solution with a mass concentration of 90-95% and refrigerated until the dehydration rate reaches 55-70%.

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