CN110326610B - Ultralow temperature cryopreservation method for sea cucumber sperms - Google Patents

Abstract

The invention discloses a cryopreservation method of sea cucumber sperms, which is sequentially carried out according to the following steps: taking sea cucumber in a breeding period, wiping off seawater of a sea cucumber body, picking out a complete gonad after dissecting from the abdomen, sucking off body fluid on the surface of the complete gonad, transferring the body fluid into a sterile culture dish, shearing into pieces, sucking semen, and filtering by using a 300-mesh bolting silk; taking the filtered semen and the frozen preservation solution according to the volume ratio of 1: 1-2, injecting the mixture into a freezing pipe, and uniformly mixing, wherein the freezing preservation solution is prepared by adding 8-15 g of glucose, 7-9 g of sodium chloride, 0.5-0.7 g of potassium chloride, 3-5 g of trehalose, 0.05-0.1 g of anhydrous calcium chloride and 80-120 mL of dimethyl sulfoxide with the purity of more than or equal to 99.7% into per liter of distilled water; and (3) suspending the freezing pipe at a position 10-15 cm away from the surface of liquid nitrogen for 10min, then suspending the freezing pipe at the surface of the liquid nitrogen for 5min, and finally immersing the freezing pipe in the liquid nitrogen for freezing and storing.

Description

Ultralow temperature cryopreservation method for sea cucumber sperms

Technical Field

The invention belongs to the technical field of cryopreservation of sea cucumber in low-temperature biology and germplasm, and particularly relates to an ultralow-temperature cryopreservation method for sea cucumber sperms.

Background

Sea cucumber belongs to Echinodermata (Echinodermata), phylum of migratory subfamily (Eleutherozea), class holothuria (Holothuroidea), is widely distributed in the temperate zone and tropical zone oceans of the world, and is the most common higher invertebrate of oceans. The sea cucumber, which is warm in nature and tonic, is rich in various nutrient elements and bioactive substances, and is a good tonic product with homology of medicine and food. With the market demand and the annual expansion of industrial scale of the sea cucumbers, excessive harvesting and catching of the wild sea cucumbers become more and more serious, so that the resource quantity and the quality of the wild sea cucumbers are sharply reduced, and the problems of low yield per unit, quality degradation and the like of the cultured sea cucumbers also occur. Therefore, researchers mostly adopt a hybridization method of north-south sea cucumbers with large geographical isolation to improve the quality of sea cucumber germplasms. However, the south-north male and female sea cucumbers are not matured synchronously, so that ripening and cleanup inducing measures are needed in the sea cucumber hybridization process, the operation is complex, the cost is high, and the problems that the hybridization cannot be realized due to ripening and cleanup inducing failure can also occur.

A paper published by Shao et al (Shao M Y, Zhang Z F, Yu L, et al. Cryopression of sea cumber 2006Apostichopus japonicus (Selenka) sperm [J]Aquaculture Research, 2006, 37(14): 1450-. The method has the operation time of 1 hour, and the sperm motility loss is large; the formula of the preservation solution is 423.00 mM NaCl, 9.00 mM KCl and 9.27 mM CaCl₂、22.94 mM MgCl₂、22.50 mM MgSO₄And 10 mM Hepes buffer + 15% dimethyl sulfoxide. The Hepes buffer solution is high in price and can generate hydrogen peroxide with certain biological toxicity when exposed to visible light; the sperm preservation time is short (1 hour), and after thawing, the sperm lifetime is only 1200s, so that the sperm preservation method is difficult to apply to production.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides an ultralow temperature cryopreservation method for sea cucumber sperms.

The technical solution of the invention is as follows: the ultralow-temperature cryopreservation method for the sea cucumber sperms is characterized by comprising the following steps of:

a. taking sea cucumber in a breeding period, wiping off seawater of a sea cucumber body, picking out a complete gonad after dissecting from the abdomen, sucking off body fluid on the surface of the complete gonad, transferring the body fluid into a sterile culture dish, shearing into pieces, sucking semen, and filtering by using a 300-mesh bolting silk;

b. taking the filtered semen and the frozen preservation solution according to the volume ratio of 1: 1-2, injecting the mixture into a freezing pipe, and uniformly mixing, wherein the freezing preservation solution is prepared by adding 8-15 g of glucose, 7-9 g of sodium chloride, 0.5-0.7 g of potassium chloride, 3-5 g of trehalose, 0.05-0.1 g of anhydrous calcium chloride and 80-120 mL of dimethyl sulfoxide with the purity of more than or equal to 99.7% into per liter of distilled water;

c. and (3) suspending the freezing pipe at a position 10-15 cm away from the surface of liquid nitrogen for 10min, then suspending the freezing pipe at the surface of the liquid nitrogen for 5min, and finally immersing the freezing pipe in the liquid nitrogen for freezing and storing.

The method has short operation time (15-20 min), and avoids the problem of great sperm motility loss; the used preservation solution has low cost and is nontoxic; the preservation time is long (17 days for sperm cryopreservation), the highest survival rate of the sperm reaches 19.00 +/-4.00%, and the service life reaches 2817.33 s; the fertilization rate of thawed and revived sperms can reach 83.72-83.74%, the cleavage rate (more than 2-4 cell periods counted) reaches 77.67-87.05%, and after fertilization is carried out for 20 hours, the blastocyst hatchability is measured to be 56.47-65.00%, so that the method is suitable for the aspects of artificial propagation, cross breeding, germplasm resource preservation and the like of sea cucumbers.

Detailed Description

a. The method is characterized in that the body length of the male sea cucumber cultured in key laboratories is 20-25 cm, and the body weight is 450-650 g. Taking out the sea cucumber from water in the breeding season of 5 months in 2019, wiping seawater on the body surface of the sea cucumber (avoiding activation of sperms by the seawater), dissecting the sea cucumber by using sterilized scissors at the abdomen, avoiding cutting off gonads, picking out complete gonads by using tweezers, sucking and drying coelomic fluid on the surfaces of the gonads by using sterile filter paper, transferring the liquid to a sterile culture dish, cutting off the gonads, sucking by using a 5ml injector, wrapping an injection port by using a 300-mesh bolting silk (high-temperature sterilization and drying at 60 ℃) for filtering, avoiding mixing the coelomic fluid and the seawater in the filtering process, collecting the filtered sperms into a 50ml centrifuge tube, and temporarily placing the centrifuge tube in an insulated box with ice blocks;

b. sucking 300 mu l of filtered semen and frozen preservation solution respectively by using a pipette, adding the filtered semen and the frozen preservation solution into a 2mL sterile frozen preservation tube, and uniformly mixing, wherein the frozen preservation solution is prepared by adding 12g of glucose, 7g of sodium chloride, 0.7g of potassium chloride, 5g of trehalose, 0.1g of anhydrous calcium chloride and 80-120 mL of dimethyl sulfoxide with the purity of more than or equal to 99.7 percent into each liter of distilled water;

c. and (3) suspending the freezing pipe at a position 15cm away from the surface of liquid nitrogen for 10min, then suspending the freezing pipe at the surface of the liquid nitrogen for 5min, and finally immersing the freezing pipe in the liquid nitrogen for freezing (-196 ℃) for preservation.

Experiment:

and (3) sperm motility detection: dipping a small amount of the semen filtered in the step a, coating the semen on a glass slide, dropwise adding a drop of seawater for activation, observing the sperm motility under a microscope, and statistically analyzing to show that the sperm motility is 85.55 +/-5.00 percent.

Thawing and detecting the activity of frozen sperms: and d, taking the freezing tube in the step c out of the liquid nitrogen, immediately putting the freezing tube into a constant-temperature water bath at 30 ℃ for unfreezing after the surface of the liquid nitrogen is balanced for 30s, and continuously shaking the freezing tube in the unfreezing process until the freezing tube is completely thawed. And (3) thawing the sperm which is frozen and preserved for 17 days, and performing sperm motility observation and statistics: smearing a small amount of thawed semen on a glass slide, dripping a drop of seawater for activation, observing the sperm motility under a microscope, and statistically analyzing to show that the sperm motility can reach 19.00 +/-4.00% at most after thawing and the service life of the sperm is 2817.33 s.

Fertilization detection after thawing and activation of frozen sperm: injecting the thawed sperm which is frozen and stored for 17 days into seawater containing the ovum (the density of the ovum is 13-26 particles/ml), and stirring the seawater up and down every 1 hour according to a conventional method to promote the combination of the sperm and the ovum to finish fertilization. After fertilization for 4 hours, washing eggs with 300-mesh bolting silk, washing off redundant sperms, and putting the fertilized eggs into seawater at 21-22 ℃ for incubation. Statistical analysis shows that the fertilization rate of thawed and revived sperms can reach 83.72-83.74%, and the cleavage rate (more than 2-4 cell stages) reaches 77.67-87.05%; after fertilization for 20 hours, the blastocyst hatchability is measured to be 56.47-65.00%.

Compared with the prior related frozen preservation solution of fish, sea urchin and shellfish: the cryopreservation liquid provided by the embodiment of the invention is used for replacing the cryopreservation liquid for the adult fish sperm, the frozen storage of the sea cucumber sperm is not changed in other steps, the activity of the thawed sea cucumber sperm is low (the highest is 11 +/-2.33%), and the life of the thawed sperm is short (539 s); the cryopreservation solution provided by the embodiment of the invention is respectively replaced by sea urchin and shellfish sperm cryopreservation solution, the sea cucumber sperm is preserved for 48 hours without changing the other steps, and the activity of the thawed sea cucumber sperm is almost zero. The cryopreservation liquid of fish, sea urchin and shellfish is not suitable for cryopreservation of sea cucumber sperms.

Claims (1)

1. The ultralow-temperature cryopreservation method for the sea cucumber sperms is characterized by comprising the following steps of:

a. taking sea cucumber in a breeding period, wiping off seawater of a sea cucumber body, picking out a complete gonad after dissecting from the abdomen, sucking off body fluid on the surface of the complete gonad, transferring the body fluid into a sterile culture dish, shearing into pieces, sucking semen, and filtering by using a 300-mesh bolting silk;

b. taking the filtered semen and the frozen preservation solution according to the volume ratio of 1: 1-2, injecting the mixture into a freezing pipe, and uniformly mixing, wherein the freezing preservation solution is prepared by adding 8-15 g of glucose, 7-9 g of sodium chloride, 0.5-0.7 g of potassium chloride, 3-5 g of trehalose, 0.05-0.1 g of anhydrous calcium chloride and 80-120 mL of dimethyl sulfoxide with the purity of more than or equal to 99.7% into per liter of distilled water;

c. and (3) suspending the freezing pipe at a position 10-15 cm away from the surface of liquid nitrogen for 10min, then suspending the freezing pipe at the surface of the liquid nitrogen for 5min, and finally immersing the freezing pipe in the liquid nitrogen for freezing and storing.