CN110326610A - Sea cucumber sperm cryopreservation method - Google Patents

Abstract

The invention discloses a sea cucumber sperm ultra-low temperature cryopreservation method, which is carried out in sequence as follows: take a sea cucumber in the breeding period and dry the sea water of the sea cucumber body, dissect the abdomen, pick out the complete gland, suck the body fluid on the surface of the complete gland, and then move it to the Cut into pieces in a sterile petri dish, absorb the semen and filter it with a 300-mesh sieve; take the filtered semen and cryopreservation solution according to the volume ratio of 1:1-2 and pour them into the cryovial to mix evenly. Add 8-15g of glucose, 7-9g of sodium chloride, 0.5-0.7g of potassium chloride, 3-5g of trehalose, 0.05-0.1g of anhydrous calcium chloride and 80-120 mL of dimethyl Sulfoxide; suspend the cryovial at a distance of 10-15 cm from the surface of liquid nitrogen for 10 minutes, then suspend the cryovial on the surface of liquid nitrogen for 5 minutes, and finally immerse the cryovial in liquid nitrogen for cryopreservation.

Description

Ultra-low temperature cryopreservation method of sea cucumber sperm

technical field

The invention belongs to the technical field of sea cucumber cryobiology and ultra-low temperature cryopreservation of germplasm, and in particular relates to a method for ultra-low temperature cryopreservation of sea cucumber sperm.

Background technique

Sea cucumbers belong to the phylum Echinodermata, the subphylum Eleutherozea, and the class Holothuroidea. They are widely distributed in temperate and tropical oceans around the world, and are the most common marine higher invertebrates. Sea cucumber "is warm and nourishing in nature, sufficient for enemy ginseng" is rich in various nutritional elements and biologically active substances, and is a good nourishing product with the same source of medicine and food. As the market demand for sea cucumbers and the scale of the industry expand year by year, overharvesting of wild sea cucumbers is becoming more and more serious, resulting in a sharp decline in the amount of resources and germplasm quality of wild sea cucumbers. Farmed sea cucumbers also have problems such as lower yields and germplasm degradation. For this reason, at present, researchers mostly adopt the hybridization method of north and south sea cucumbers with large geographical isolation to improve the quality of sea cucumber germplasm. However, because the male and female sea cucumbers of the north and the south do not mature synchronously, so that in the process of sea cucumber hybridization, methods such as accelerated ripening and excretion promotion are required. Unable to achieve and other issues.

In 2006, the paper published by Shao et al. (Shao MY , Zhang ZF , Yu L , et al.Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm [J].Aquaculture Research, 2006, 37(14):1450-1457. ) was used to dilute the sea cucumber sperm with the preservation solution and then cryopreserved. The operation time of this method is up to 1 hour, and the sperm motility is greatly lost; the preservation solution formula is 423.00 mM NaCl, 9.00 mM KCl, 9.27 mM CaCl ₂ , 22.94 mM MgCl ₂ , 22.50 mM MgSO ₄ and 10 mM Hepes buffer + 15% dimethylformaldehyde sulfoxide. Hepes buffer is not only expensive but also produces hydrogen peroxide with certain biological toxicity when exposed to visible light; the storage time of sperm is short (1 hour), and after thawing, the life span of sperm is only 1200s, which is difficult to apply to production.

Contents of the invention

The present invention aims to solve the above-mentioned technical problems existing in the prior art, and provides a method for cryopreserving sea cucumber sperm at ultra-low temperature.

The technical solution of the present invention is: a kind of sea cucumber sperm cryopreservation method, it is characterized in that follow the following steps successively:

a. Take the sea cucumbers in the breeding season and dry the sea water of the ginseng body. After dissecting the abdomen, pick out the complete glands, blot up the body fluids on the surface of the complete glands, move them to a sterile petri dish, cut them into pieces, absorb the semen and filter them with a 300-mesh sieve ;

b. Take the filtered semen and cryopreservation solution and inject them into the cryopreservation tube according to the volume ratio of 1:1~2 and mix evenly. The cryopreservation solution is added with 8~15g of glucose and 7~9g of sodium chloride per liter of distilled water , 0.5-0.7g potassium chloride, 3-5g trehalose, 0.05-0.1g anhydrous calcium chloride and 80-120 mL dimethyl sulfoxide with a purity ≥99.7%;

c. Suspend the cryovial at a distance of 10-15 cm from the surface of liquid nitrogen for 10 minutes, then suspend the cryovial on the surface of liquid nitrogen for 5 minutes, and finally immerse the cryovial in liquid nitrogen for cryopreservation.

The present invention has a short operation time (15-20 min) and avoids the problem of large loss of sperm motility; the preservation solution used is low-cost and non-toxic; the preservation time is long (17 days for sperm cryopreservation), and the highest sperm survival rate reaches 19.00±4.00 %, the lifespan reaches 2817.33s; the fertilization rate of thawed and recovered sperm can reach 83.72-83.74%, the cleavage rate (statistically 2-4 cell stage and above) reaches 77.67-87.05%, and after 20 hours of fertilization, the blastocyst hatching rate is determined 56.47-65.00%, suitable for sea cucumber artificial propagation, hybrid breeding, and germplasm resource preservation.

Detailed ways

a. Male sea cucumbers cultivated by the Key Laboratory of Northern Seawater Enrichment and Aquaculture of the Department of Agriculture and Rural Affairs, Dalian Ocean University, with a body length of 20-25cm and a weight of 450-650g. During the breeding season in May 2019, take the sea cucumber out of the water and dry the surface of the sea cucumber (to avoid seawater to activate sperm), dissect the sea cucumber abdomen with sterilized scissors, avoid cutting the gonads, and pick out the complete glands with tweezers , use sterile filter paper to blot the body cavity fluid on the surface of the gonads, transfer them to a sterile petri dish, cut the gonads into pieces, draw them with a 5ml syringe, and wrap the injection port with a 300-mesh sieve (sterilized at high temperature and dried at 60°C) Carry out filtration, avoid body cavity fluid and seawater mixing during the filtration process, collect the filtered sperm into a 50ml centrifuge tube, and temporarily place it in an incubator with ice cubes;

b. Use a pipette gun to absorb 300 μl of the filtered semen and cryopreservation solution, add them into a 2ml sterile cryopreservation tube and mix evenly. The cryopreservation solution is 12 g of glucose, 7 g of sodium chloride, and 0.7 g of Potassium chloride, 5g trehalose, 0.1g anhydrous calcium chloride and 80-120 mL dimethyl sulfoxide with a purity ≥99.7%;

c. Suspend the cryovial at a distance of 15cm from the surface of liquid nitrogen for 10 minutes, then suspend the cryovial on the surface of liquid nitrogen for 5 minutes, and finally immerse the cryovial in liquid nitrogen to freeze (-196°C) for storage.

experiment:

Sperm motility test: dip a small amount of semen filtered in step a and spread it on a glass slide, add a drop of seawater to activate it, and observe the sperm motility under a microscope. Statistical analysis shows that the sperm motility is 85.55±5.00%.

Thawing and motility testing of frozen sperm: take the cryovial in step c out of liquid nitrogen, equilibrate on the surface of liquid nitrogen for 30 seconds, and immediately thaw it in a constant temperature water bath at 30°C. During the thawing process, shake the cryovial continuously until it is completely thawed. Thaw frozen and preserved sperm for 17 days, observe and count sperm motility: take a small amount of thawed semen and smear it on a glass slide, add a drop of seawater to activate it, and observe the sperm motility under a microscope. Statistical analysis shows that the sperm motility after thawing The highest can reach 19.00±4.00%, and the life span is 2817.33s.

Fertilization detection after thawing and activation of frozen sperm: Inject the thawed sperm frozen for 17 days into the seawater containing eggs (the density of eggs is 13-26/ml), and stir the seawater up and down every 1 hour according to the conventional method to promote the fertilization of sperm and eggs. Conjugation completes fertilization. After 4 hours of fertilization, wash the eggs with a 300-mesh sieve to remove excess sperm, and place the fertilized eggs in seawater at 21-22°C for incubation. Statistical analysis shows that the fertilization rate of thawed and revived sperm can reach 83.72-83.74%, and the cleavage rate (statistically above 2-4 cell stage) can reach 77.67-87.05%; after 20 hours of fertilization, the measured blastocyst hatching rate is 56.47-65.00 %.

Compared with the relevant cryopreservation solutions of existing fish, sea urchins and shellfish: the cryopreservation solution of the embodiment of the present invention is replaced with the fish sperm cryopreservation solution, and other steps remain unchanged and the sea cucumber sperm are frozen for 48 hours. After thawing, the sea cucumber sperm Low motility (maximum: 11±2.33%) and short lifespan of sperm after thawing (539s); the cryopreservation solution of the embodiment of the present invention was replaced with sea urchin and shellfish sperm cryopreservation solution, and other steps remained unchanged. After thawing, the motility of sea cucumber sperm is almost zero. It shows that the cryopreservation solution of fish, sea urchin and shellfish is not suitable for the cryopreservation of sea cucumber sperm.

Claims (1)

1. a sea cucumber sperm cryopreservation method is characterized in that following the steps successively: a. Take the sea cucumbers in the breeding period and dry the sea water of the ginseng body. After dissecting the abdomen, pick out the integrity glands, blot up the body fluids on the surface of the integrity glands, move them to a sterile petri dish, cut them into pieces, absorb the semen and filter them with a 300-mesh sieve ; b. Take the filtered semen and the cryopreservation solution and inject them into the cryopreservation tube according to the volume ratio of 1:1~2 and mix them evenly. , 0.5-0.7g potassium chloride, 3-5g trehalose, 0.05-0.1g anhydrous calcium chloride and 80-120 mL dimethyl sulfoxide with a purity ≥99.7%; c. Suspend the cryovial at a distance of 10-15 cm from the surface of liquid nitrogen for 10 minutes, then suspend the cryovial on the surface of liquid nitrogen for 5 minutes, and finally immerse the cryovial in liquid nitrogen for cryopreservation.