CN114258911B - Cryopreservation solution and cryopreservation method for sparus latus sperms - Google Patents
Cryopreservation solution and cryopreservation method for sparus latus sperms Download PDFInfo
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Abstract
The invention discloses a cryopreservation solution for sparus latus sperms, which comprises a sperm base diluent, nutrient substances, an antioxidant and an anti-freezing protective agent; the sperm basic diluent is formed by mixing a cortland solution and a Hank's solution; the nutrient substances comprise sucrose and trehalose; the antioxidant comprises bovine serum albumin, melatonin and vitamin E; the anti-freezing protective agent is dimethyl sulfoxide. The cryopreservation solution has remarkable advantages when being applied to cryopreservation of the yellow fin porgy sperms, so that the survival rate and the activity of the frozen preserved yellow fin porgy sperms are high. The invention also discloses a cryopreservation method of the sparus latus sperms.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cryopreservation solution and a cryopreservation method for sperms of yellow-fin sparus.
Background
The yellow-fin Pagrus macrocephalus belongs to Pagrus, genus acanthopagrus, is a seawater fish with wide salinity adaptability, and is mainly distributed in southeast Asia coastal sea fish and estuary; in the breeding season, the yellow fin sea bream migrates from a low salinity shallow sea area to a high salinity sea area to lay eggs. The yellow-fin sea bream is a natural hermaphrodite fish: sex of 1-age yellow fin sea bream is full male, after 1-age male passes in the breeding period, the spermary of 1-age male gradually degenerates, the ovary begins to develop, the yellow fin sea bream which develops to 2-age is hermaphrodite and can generate sperms, but the ovum is immature; part of the 3-year-old yellow fin sea bream is completely turned into female sex, and mature ovum can be generated.
The yellow-fin sparus is very popular in the market due to delicious meat, higher nutritional value and good taste, and is widely cultivated in the southeast coastal areas of China. In recent years, the culture scale of the yellow-fin snapper is gradually increased, the demands of farmers on high-quality yellow-fin snapper seedlings are increased, the number of sea-caught yellow-fin snapper seedlings is difficult to meet the demands, and the sea-caught yellow-fin snapper seedlings have uneven quality, so that the cultivation of a new high-quality yellow-fin snapper variety has important significance for improving the culture efficiency and social and economic benefits. However, as described above, the maturation time of the sparus latus parent is long and the maturation time of the parent is inconsistent, which makes it difficult to breed the sparus latus fine breed. Therefore, the method has important significance for cultivating new varieties of the excellent sparus latus by freezing and storing the excellent germ plasm resources of the sparus latus and applying the resources to artificial propagation.
Germplasm resources, commonly referred to as embryos, embryonic stem cells, and germ cells, i.e., germ cells, are the basis for the multiplication of species or populations. In most sexual animals, germ cells undergo meiosis to produce sperm and eggs, which combine to form a fertilized egg to produce a new individual. Most fish embryos are developed in vitro, and energy required for early development is derived from a large amount of nutrients such as yolk and protein carried by mature eggs. For the preservation of germplasm resources, the mature eggs of fishes are large and are not beneficial to permeation and balance, and substances such as a large amount of yolk, protein and the like contained in the eggs are difficult to permeate and balance during freezing and other lipid substances and are easy to inactivate during freezing and thawing, so that the freezing preservation of the eggs of the fishes is difficult. And most of cytoplasmic inclusions can be gradually removed in the process of the mature sperms, and the mature sperms have the characteristics of small volume, large quantity, relatively simple inclusion substances and the like, so the sperms are an excellent choice for storing fish germplasm resources.
At present, the low-temperature cryopreservation technology is primarily applied to cryopreservation of sperms of some seawater fishes, such as rainbow trout, red drum fish, Russian sturgeon and the like. For the low-temperature cryopreservation technology of sperms, the sperm cryopreservation liquid is the key for cryopreservation of the sperms. In the prior art, the sperm cryopreservation liquid for fish forms a solution environment similar to a physiological state through various salt ions to temporarily maintain the normal physiological state of the sperm; in addition, the sperm cryopreservation liquid is added with an antifreeze (glycerol, DMSO, methanol and the like) and a non-permeable antifreeze or nutrient substance (skimmed milk powder, egg yolk, protein, sugar and the like) to protect the integrity and the vitality of the internal structure of the sperm during the process of drastic temperature change.
Regarding the sperm of the yellow fin sea bream, the ultrastructure shows that the head of the sperm is round or oval, the head has no obvious acrosome structure, the implantation pit is positioned in the middle of the bottom end of the cell nucleus, the center particle complex is positioned in the implantation pit, the center particle at the near end is positioned at the upper section of the implantation pit, and the substrate is positioned at the lower section of the implantation pit; the oversleeve is positioned at the rear end of the nucleus, and mitochondria and vesicles are distributed on two sides of the oversleeve; the tail part is slender, the main structure of the tail part is a shaft wire, and the shaft wire is a typical '9 + 2' microtubule structure.
As a marine fish which naturally reverses sex from male to female, the sparus latus has a significant difference from mammals and some other marine fishes in the physiological structure of sperm. First, in comparison to mammals, the yellow fin bream has no typical acrosome structure; compared with the seawater fish epinephelus coioides which naturally reverses from female to male, the sparus latus sperms have no larger gaps and a small amount of vesicles between the plasma membrane and the nuclear membrane, and do not like the goldfish sperms with a plurality of vacuoles between the nuclear membrane and the plasma membrane; unlike the situation that the nest for implanting sperm of carp is positioned at one side of nucleus and the nest for implanting sperm of large yellow croaker is positioned at the back of nucleus, the sperm implanting nests of yellow fin sea bream, Japanese eel, flat sea bream and pelteobagrus fulvidraco are similar to the sperm implanting nests of Japanese eel, flat sea bream and pelteobagrus fulvidraco, and are sunk into the nucleus from the back end to the front end of the nucleus to form a 'well' shape; the cytoplasmic membrane of the sperm of the yellow-fin porgy is not connected with the oversleeve membrane, which is different from the sperm of Japanese eel; no other obvious structure is found on the tail of the yellow-fin sea bream sperm except the axial filament, and the lateral fin structure of the tail of the sperm such as flat sea bream, red-fin takifugu and the like, and the basket-shaped structure of the tail of the large yellow croaker sperm are not present. As described above, there is a significant difference in the physiological structure between the sperm of the yellow fin bream and the sperm of mammals and many fishes, and therefore, the conventional fish sperm cryopreservation liquid is not optimal for the sperm of the yellow fin bream and cannot guarantee a high survival rate and vitality after cryopreservation. Therefore, there is a need to develop a sperm cryopreservation solution for sparus latus sperm, which is specific to the characteristics of sparus latus sperm.
Disclosure of Invention
The first purpose of the invention is to provide a cryopreservation solution for the sperms of the oplegnathus fasciatus, which is optimized and improved on the basis of the existing cryopreservation solution for the sperms of the fishes, so that the sperms of the oplegnathus fasciatus have higher survival rate and activity after cryopreservation.
The second object of the present invention is to provide a method for cryopreserving sparus latus sperm using the cryopreservation solution.
In order to achieve the first object, the invention adopts the technical scheme that:
a cryopreservation solution for Sparus latus sperm comprises sperm base diluent, nutrients, antioxidant and antifreeze protectant; the sperm basic diluent is formed by mixing a corrland solution and a Hank's solution so as to ensure that the sperms of the yellow fin sea bream obtain higher activity; the nutrient substances comprise sucrose and trehalose; the antioxidant comprises Bovine Serum Albumin (BSA), melatonin and vitamin E; the anti-freezing protective agent is dimethyl sulfoxide (DMSO).
Specifically, the cortland solution comprises the following components: sodium chloride, sodium dihydrogen phosphate, sodium bicarbonate, potassium chloride, calcium chloride dihydrate, D-glucose and magnesium sulfate heptahydrate; the Hank's solution comprises the following components: sodium chloride, disodium hydrogen phosphate, sodium bicarbonate, potassium dihydrogen phosphate, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, D-glucose and magnesium sulfate heptahydrate.
The inventor finds that the survival rate of the sparus latus sperms after cryopreservation is low and the vitality is low when the existing fish sperm cryopreservation liquid is used for cryopreservation of the sparus latus sperms; it is found that in the cooling balance stage before semen cryopreservation, sperm can generate a large amount of active oxygen due to respiration to cause sperm plasma membrane damage, and the cryopreservation effect of the sperm of the oplegnathus fasciatus is influenced.
Therefore, the invention introduces the antioxidant component into the cryopreservation solution of the yellow fin sea bream sperms; then, through a large number of experiments, the inventor obtains the antioxidant combination of bovine serum albumin, melatonin and vitamin E, and the antioxidant combination is used for cryopreservation of the yellow fin sea bream sperms, so that the survival rate and the activity of the frozen yellow fin sea bream sperms are obviously improved.
Wherein, the melatonin is firstly applied to sperm cryopreservation solution of fish; previously, research shows that in bulls, the addition of melatonin can obviously improve the proportion of linearly moving sperms, improve the activity of the sperms, increase the total protein of seminal plasma, the concentration of albumin and cholesterol, and reduce the activity of aspartate aminotransferase; in hamsters, melatonin can remarkably promote the hyperactivation of sperms, so as to enhance the in vitro fertilization capability of the sperms; therefore, melatonin is used as an antioxidant in the prior art for cryopreservation fluids to protect mammalian sperm. However, unlike mammals, most fish sperm (including yellowfin sea bream sperm) have no acrosome structure, and physiological structures such as sperm head inclusion are also significantly different from those of the mammal sperm, so that the cryopreservation solution for the mammal sperm is not suitable for cryopreservation of the fish sperm.
However, through a large number of studies, experiments carried out by adding melatonin into a cryopreservation solution of the sperm of the yellow fin sea bream prove that the melatonin can be applied to the cryopreservation solution of the sperm of the yellow fin sea bream and can effectively improve the survival rate and the activity of the sperm of the yellow fin sea bream after cryopreservation.
In addition, the inventor also innovatively applies the vitamin E to the yellow fin porgy sperm cryopreservation liquid which is matched with the melatonin, so that the survival rate and the activity of the yellow fin porgy sperm after cryopreservation are obviously improved.
In addition, in the process of researching the cryopreservation solution of the sperm of the yellow fin sea bream, the inventor considers that the pH value of the optimal sperm motility of different fishes is different, for example, the sperm motility and the fertilization rate of rainbow trout are highest at the pH value of 9.0, the sperm motility of halibut and lateolabrax is highest at the pH value of 7.5-8.5 and the sperm motility is highest at the pH value of 9.0, the sperm motility of epinephelus coioides is best at the pH value of 8.4, and the sperm motility of the yellow fin sea bream is best at the pH value of 7.8-8.0, so the inventor takes the pH value of the cryopreservation solution into one of the important factors influencing the sperm motility of the frozen yellow fin sea bream. Therefore, based on that the sperm motility of the yellow fin sea bream is best when the pH value is 7.8-8.0, the inventor optimizes the existing fish sperm cryopreservation solution, selects a corrland solution and a Hank's solution which have the pH value range close to the range of the sperm motility of the yellow fin sea bream which is the best, and finds that when the two solutions are mixed as a sperm basic diluent to be used in the cryopreservation solution of the sperm of the yellow fin sea bream, the survival rate is ensured, and the sperm of the yellow fin sea bream can be ensured to obtain higher motility.
The preferred scheme of the invention is as follows:
in the sperm base diluent, the volume ratio of a Cortland solution to a Hank's solution is 2: 1-1: 3; preferably, the Cortland solution and the Hank's solution are mixed in a volume ratio of 1: 2.
In the sparus latus sperm cryopreservation solution, the addition amount of trehalose is 35.0-45.0 g/L and the addition amount of sucrose is 6-7 g/L on the basis of a sperm base diluent; preferably, the addition amounts of trehalose and sucrose are 38.7g/L and 6.85g/L, respectively.
In the sparus latus sperm cryopreservation solution, the sperm base diluent is taken as a reference, the mass percentage addition amount of Bovine Serum Albumin (BSA) is 1-3%, the addition amount of melatonin is 1-100 mu mol/L, and the addition amount of vitamin E is 1-10 mu mol/L; preferably, the addition amount of BSA is 2% by mass, the addition amount of melatonin is 10. mu. mol/L, and the addition amount of vitamin E is 5. mu. mol/L.
In the cryopreservation solution for the sperms of the yellow fin sea bream, the addition amount of dimethyl sulfoxide (DMSO) in percentage by mass is 5-15% based on the basic diluent of the sperms; preferably, the DMSO is added in an amount of 10% by mass.
The cryopreservation solution for the sperms of the yellow fin sea bream can be applied to the cryopreservation technology of the sperms of the yellow fin sea bream.
In order to achieve the second object, the invention adopts the technical scheme that:
a sparus latus sperm cryopreservation method using the cryopreservation solution comprises the following steps:
s1, semen collection: in the breeding season of the sparus latus (about 10 months), selecting healthy male parent fish with mature spermary, wiping water around cloaca holes of a fish body with a clean towel, lightly pressing the abdomen until the cloaca holes flow out milky semen, and collecting the semen with a 1ml centrifugal tube;
s2, preparing the sparus latus sperm cryopreservation solution;
s3, freezing and storing sperm: uniformly mixing the sparus latus sperm cryopreservation solution and the semen in a cryopreservation tube, and diluting the semen; firstly, placing a freezing tube on ice, incubating and keeping the freezing tube away from light for 10-30 minutes, then placing the tube above the surface of liquid nitrogen for freezing and staying for 10-30 minutes, then immersing the tube in the liquid nitrogen for rapid freezing treatment, and finally transferring the tube to a low temperature of-150 ℃ for preservation;
s4, sperm recovery: after the sperm are frozen for a period of time, taking out the cryopreservation tube filled with the sperm from liquid nitrogen, then immediately and rapidly thawing in a 37 ℃ water bath, and preserving the thawed sperm at 0-4 ℃;
s5, sperm survival rate detection: diluting the thawed sperms with a sperm basic diluent, dyeing the sperms with trypan blue staining solution, and observing under a microscope to evaluate the survival rate of the sperms; secondly, diluting the thawed sperms with a sperm basic dilution solution, and observing the sperms respectively with Propidium Iodide (PI) and 4', 6-diamidino-2-phenylindole (DAPI) under a fluorescence microscope to count the survival rate of the sperms;
s6, sperm motility detection: and taking the thawed semen, activating the sperm, taking a small amount of sperm solution, observing under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm solution.
In the step S1, after semen is collected, to determine the concentration and the survival rate of the collected fresh semen, the collected semen is diluted by 10 times with a sperm-based dilution solution, and cell viability detection and cell counting plate counting are performed; after cell counting and survival rate detection, selecting the sperm with the survival rate of more than 98 percent and the sperm concentration of 6 multiplied by 108The semen with the volume of more than one/ml is subjected to subsequent operations.
Compared with the prior art, the invention has the beneficial effects that:
the cryopreservation solution for the sperms of the yellow fin sea bream provided by the invention has remarkable advantages when being applied to cryopreservation of the sperms of the yellow fin sea bream, and ensures that the survival rate and the vitality of the sperms of the yellow fin sea bream after cryopreservation are at a higher level, namely the survival rate of the thawed sperms reaches more than 90 percent and the vitality reaches more than 49 percent;
according to the invention, by improving the sperm base diluent and optimizing the antioxidant component in the cryopreservation solution, particularly adding melatonin and vitamin E, the survival rate and activity of the cryopreserved sparus latus sperm are obviously improved; the cryopreservation solution for the sperms of the sparus latus can be used for protecting sparus latus germplasm resources and carrying out large-scale artificial propagation and production, and has important significance for sparus latus germplasm resource protection and genetic improvement.
Detailed Description
The following examples are only for illustrating the present invention, and the scope of the present invention is not limited to only the following examples. The objectives of the present invention can be achieved by the ordinary skilled person in the art according to the disclosure of the present invention and the ranges of the parameters.
The preparation method of the cryopreservation solution for the yellow fin sea bream sperms in the specific embodiment of the invention comprises the following steps:
s1, preparing a sperm basic diluent: preparing cottland solution and Hank's solution according to the proportion, blending the pH values, and mixing the cottland solution and the Hank's solution according to the proportion to obtain a sperm basic diluent;
and S2, sequentially adding the nutrient substances, the antioxidant and the antifreeze into the sperm basic diluent according to the mass concentration or molar concentration, uniformly mixing, filtering by a 0.22-micron filter membrane to obtain sterile frozen stock solution, and storing at 4 ℃ for a long time.
Wherein the components of the cortland solution and the Hank's solution are respectively as follows:
cortland solution: 7.66g/L sodium chloride, 0.51g/L sodium dihydrogen phosphate, 1.16g/L sodium bicarbonate, 0.38g/L potassium chloride, 0.31g/L calcium chloride dihydrate, 1.00g/L D-glucose, 0.26g/L magnesium sulfate heptahydrate, pH 7.0.
Hank's solution: 8.07g/L sodium chloride, 0.048g/L disodium hydrogen phosphate, 0.35g/L sodium hydrogen carbonate, 0.060g/L potassium dihydrogen phosphate, 0.40g/L potassium chloride, 0.19g/L calcium chloride dihydrate, 0.10g/L magnesium chloride hexahydrate, 1.00g/L D-glucose, and 0.11g/L magnesium sulfate heptahydrate, pH 7.5.
The invention is used for collecting the seminal fluid before the preservation of the sperm of the sparus latus by freezing, and the operation is as follows:
in the breeding season of the sparus latus, namely 10 months, healthy male parent fishes with mature spermary development of different individuals are selected, a clean towel is used for wiping water around cloacal holes of the fish bodies, milky semen flows out from the cloacal holes by lightly pressing the abdomen, and 10 groups of semen are collected by a 1ml centrifugal tube.
For the collected semen, the sperm survival rate and the sperm concentration are firstly confirmed, and the sperm survival rate and the sperm concentration are specifically as follows: diluting 10 groups of semen with sperm-based diluent solution (Cortland solution: Hank's solution: 2:3) by 10 times, detecting cell activity and counting with cell counter plate to confirm that semen survival rate is above 98% and semen concentration is 6 × 108And (4) performing subsequent cryopreservation operation after the culture medium is more than one per ml.
Example 1
(1) Preparing a sparus latus sperm cryopreservation solution 1: mixing the Cortland solution and the Hank's solution according to the volume ratio of 1:2 to prepare a sperm basic diluent solution 1; in the sperm base stock solution 1, based on the above, 38.7g/L trehalose, 6.85g/L sucrose, 2% (wt%) BSA, 10. mu. mol/L melatonin, 5. mu. mol/L vitamin E, and 10% (wt%) DMSO were added to obtain a sparus latus sperm cryopreservation solution 1.
(2) Freezing and storing sperms: mixing the sparus latus sperm cryopreservation solution 1 and 10 groups of semen in a cryopreservation tube according to the volume of 2:1, incubating for 20 minutes on ice, transferring to a place 4 cm above liquid nitrogen, staying for 15 minutes, and immersing the cryopreservation tube in liquid nitrogen for preservation.
(3) Thawing and recovering sperms: after the sperm are frozen for 11 months, the freezing tube filled with the sperm is taken out of a refrigerator at the temperature of-150 ℃, then immediately unfrozen in water bath at the temperature of 37 ℃ by rapid and gentle shaking, and immediately placed on ice (at the temperature of 0-4 ℃) for dark storage after unfreezing.
(4) And (3) sperm survival rate detection: the thawed semen is diluted with a sperm-based dilution solution 1, and the number of dead sperm stained with PI and fluorescent in red and all sperm stained with DAPI and fluorescent in blue is counted, respectively, with Propidium Iodide (PI) at 10. mu.g/ml and 4', 6-diamidino-2-phenylindole (DAPI) at 10. mu.g/ml, observed under a fluorescence microscope, to calculate the proportion of viable sperm. And secondly, diluting the thawed sperms by using a sperm basic dilution solution 1, staining the sperms by using 1% trypan blue staining solution and activated sperms, observing the sperms under a microscope, and evaluating the survival rate of the sperms again. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after 11 months of cryopreservation in the yellow fin sea bream sperm cryopreservation solution 1, the survival rate of 10 groups of yellow fin sea bream sperms is (4010/4164, 96.3%), and the specific results are shown in table 1.
(5) And (3) sperm motility detection: and (3) taking the thawed semen, activating the sperm by using sterilized seawater with the same volume of 25 per mill, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm was the total sperm. After 11 months of cryopreservation in the yellow fin bream sperm cryopreservation solution 2, the motility of the yellow fin bream sperm in 10 groups was (431/745, 57.8%), and the specific results are shown in table 1.
Example 2
(1) Preparing a sparus latus sperm cryopreservation solution 2: the Cortland solution and the Hank's solution were mixed at a volume ratio of 1:1 to prepare a sperm-based dilution solution 2. In the sperm basic dilution solution 1, based on the above, 35g/L of trehalose, 6g/L of sucrose, 1% (wt%) of BSA, 1. mu. mol/L of melatonin, 1. mu. mol/L of vitamin E, and 5% (wt%) of DMSO were added to obtain a sparus latus sperm cryopreservation solution 2.
(2) Freezing and storing sperms: mixing the sparus latus sperm cryopreservation solution 2 and the collected 10 groups of sperms in a cryopreservation tube according to the volume of 2:1, incubating for 20 minutes on ice, transferring to a place 5 cm above liquid nitrogen, staying for 15 minutes, and immersing the cryopreservation tube in liquid nitrogen for preservation.
(3) And (3) thawing and recovering sperms: after the sperm are frozen for 11 months, the freezing tube filled with the sperm is taken out of a refrigerator at the temperature of-150 ℃, then immediately unfrozen in water bath at the temperature of 37 ℃ by rapid and gentle shaking, and immediately placed on ice (at the temperature of 0-4 ℃) for dark storage after unfreezing.
(4) And (3) sperm survival rate detection: the thawed semen is diluted with a sperm base dilution solution 2, and observed under a fluorescence microscope using 10. mu.g/ml of Propidium Iodide (PI) and 10. mu.g/ml of 4', 6-diamidino-2-phenylindole (DAPI), the number of dead sperm stained with PI in reddish fluorescent color and all sperm stained with DAPI in bluish fluorescent color are counted, respectively, to calculate the ratio of viable sperm. And secondly, diluting the thawed sperms by using a sperm basic diluent solution 2, staining the sperms by using 1% trypan blue staining solution and activated sperms, and observing the sperms under a microscope to evaluate the survival rate of the sperms. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after 11 months of cryopreservation in the yellow fin sea bream sperm cryopreservation solution 2, the survival rate of 10 groups of yellow fin sea bream sperms is (3575/3924, 91.2%), and the specific results are shown in table 1.
(5) And (3) sperm motility detection: and (3) taking the thawed semen, activating the sperm by using sterilized seawater with the same volume of 25 per mill, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm to total sperm. After 11 months of cryopreservation in the yellow fin bream sperm cryopreservation solution 2, the motility of the yellow fin bream sperm in 10 groups was (340/693, 49.1%), and the specific results are shown in table 1.
Example 3
(1) Preparing a sparus latus sperm cryopreservation solution 3: the Cortland solution and the Hank's solution were mixed at a volume ratio of 1:3 to prepare a sperm-based dilution solution 3. In the sperm basic dilution solution 3, based on the above, 38.7g/L trehalose, 7g/L sucrose, 2% (wt%) BSA, 8 μmol/L melatonin, 7 μmol/L vitamin E, and 8% (wt%) DMSO were added to obtain a yellow sea bream sperm cryopreservation solution 3.
(2) Freezing and storing sperms: mixing the sparus latus sperm cryopreservation solution 3 and 10 groups of sperms in a cryopreservation tube according to the volume of 2:1, incubating on ice for 20 minutes, transferring to a place 5 cm above liquid nitrogen, staying for 15 minutes, and immersing the cryopreservation tube in liquid nitrogen for preservation.
(3) Thawing and recovering sperms: after the sperm are frozen for 11 months, the freezing tube filled with the sperm is taken out of a refrigerator at the temperature of-150 ℃, then immediately unfrozen in water bath at the temperature of 37 ℃ by rapid and gentle shaking, and immediately placed on ice (at the temperature of 0-4 ℃) for dark storage after unfreezing.
(4) And (3) sperm survival rate detection: the thawed semen is diluted with a sperm base diluent solution 3, and observed under a fluorescence microscope using 10. mu.g/ml of Propidium Iodide (PI) and 10. mu.g/ml of 4', 6-diamidino-2-phenylindole (DAPI), the number of dead sperm stained with PI in reddish fluorescent color and all sperm stained with DAPI in bluish fluorescent color are counted, respectively, to calculate the ratio of viable sperm. And secondly, diluting the thawed sperms by using a basic dilution solution, staining the sperms by using 1% trypan blue staining solution and activated sperms, and observing the sperms under a microscope to evaluate the survival rate of the sperms. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after the red snapper sperms are frozen in the red snapper sperm freezing preservation solution 3 for 11 months, the survival rate of 10 red snapper sperms is (3924/4117, 95.4%), and the specific results are shown in table 1.
(5) And (3) sperm motility detection: and (3) taking the thawed semen, activating the sperm by using sterilized seawater with the same volume of 25 per mill, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm to total sperm. After 11 months of cryopreservation in the yellow fin bream sperm cryopreservation solution 3, the motility of the yellow fin bream sperm in 10 groups was (398/721, 55.2%), and the specific results are shown in table 1.
Example 4
(1) Preparing a sparus latus sperm cryopreservation solution 4: the Cortland solution and the Hank's solution were mixed at a ratio of 1:2 to prepare a sperm cell base diluent solution 4. In the sperm basic dilution solution 4, based on the above, 45g/L trehalose, 6.85g/L sucrose, 3% (wt%) BSA, 100. mu. mol/L melatonin, 10. mu. mol/L vitamin E, and 10% (wt%) DMSO were added to obtain a yellow sea bream sperm cryopreservation solution 4.
(2) Freezing and storing sperms: mixing the sparus latus sperm cryopreservation solution 4 and 10 groups of sperms in a cryopreservation tube according to the volume of 2:1, incubating for 20 minutes on ice, transferring to a place 4 cm above liquid nitrogen, staying for 15 minutes, and immersing the cryopreservation tube in liquid nitrogen for preservation.
(3) Thawing and recovering sperms: after the sperm are frozen for 11 months, the freezing tube filled with the sperm is taken out of a refrigerator at the temperature of-150 ℃, then immediately unfrozen in water bath at the temperature of 37 ℃ by rapid and gentle shaking, and immediately placed on ice (at the temperature of 0-4 ℃) for dark storage after unfreezing.
(4) And (3) sperm survival rate detection: the thawed semen is diluted with a sperm-based dilution solution 4, and the number of dead sperm stained with PI and fluorescent in red and all sperm stained with DAPI and fluorescent in blue is counted, respectively, with Propidium Iodide (PI) at 10. mu.g/ml and 4', 6-diamidino-2-phenylindole (DAPI) at 10. mu.g/ml, observed under a fluorescence microscope, to calculate the proportion of viable sperm. And secondly, diluting the thawed sperms by using a sperm basic diluent solution 4, staining the sperms by using 1% trypan blue staining solution and activated sperms, and observing the sperms under a microscope to evaluate the survival rate of the sperms. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after 11 months of cryopreservation in the yellow fin sea bream sperm cryopreservation solution 4, the survival rate of 10 groups of yellow fin sea bream sperms is (3703/3965, 93.4%), and the specific results are shown in table 1.
(5) And (3) sperm motility detection: taking the thawed semen, activating the sperm with sterilized seawater of 25 per mill equal volume, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm to total sperm. After 11 months of cryopreservation in the yellow fin bream sperm cryopreservation solution 4, the motility of the yellow fin bream sperm in 10 groups was (364/685, 53.2%), and the specific results are shown in table 1.
Comparative example 1
(1) Preparation of control cryopreservation solution 1: control sperm base dilution solution 1 was prepared by mixing Cortland solution and Hank's solution at a volume ratio of 1: 2. In control sperm cell base dilution solution 1, based on this, trehalose 38.7g/L, sucrose 6.85g/L, 2% (wt%) BSA, 10% (wt%) DMSO were added, and control cryopreservation solution 1 was finally obtained.
(2) Freezing and storing sperms: the control cryopreservation solution 1 and 10 groups of sperms are mixed uniformly in a cryopreservation tube according to the volume of 2:1, incubated on ice for 20 minutes, transferred to a place 4 cm above liquid nitrogen for staying for 15 minutes, and then the cryopreservation tube is immersed in liquid nitrogen for preservation.
(3) Thawing and recovering sperms: after the sperm are frozen for 11 months, the freezing tube filled with the sperm is taken out of a refrigerator at the temperature of-150 ℃, then immediately unfrozen in water bath at the temperature of 37 ℃ by rapid and gentle shaking, and immediately placed on ice (at the temperature of 0-4 ℃) for dark storage after unfreezing.
(4) And (3) sperm survival rate detection: the number of dead sperm stained with PI in reddish fluorescent color and all sperm stained with DAPI in bluish fluorescent color were counted separately by diluting thawed semen with control sperm base dilution solution 1, and observing with Propidium Iodide (PI) at 10. mu.g/ml and 4', 6-diamidino-2-phenylindole (DAPI) at 10. mu.g/ml under a fluorescence microscope to calculate the ratio of viable sperm. And secondly, diluting the thawed sperms by using a contrast sperm basic dilution solution 1, staining the sperms by using 1% trypan blue staining solution and activated sperms, and observing the sperms under a microscope to evaluate the survival rate of the sperms. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after 11 months of frozen preservation in the control frozen preservation solution 1, the survival rate of 10 groups of sparus latus sperm is (3577/4189, 85.4%), and the specific results are shown in table 1.
(5) And (3) sperm motility detection: and (3) taking the thawed semen, activating the sperm by using sterilized seawater with the same volume of 25 per mill, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm to total sperm. After 11 months of cryopreservation in the control cryopreservation solution 1, the motility of the sperm of the 10 groups of the yellow fin sea bream was (369/895, 41.2%), and the specific results are shown in table 1.
Comparative example 2
(1) Preparation of control cryopreservation solution 2: control sperm base dilution solution 2 was prepared by mixing Cortland solution and Hank's solution at a volume ratio of 1: 2. In control sperm cell base dilution solution 2, based thereon, trehalose 38.7g/L, sucrose 6.85g/L, 2% (wt%) BSA, 5. mu. mol/L vitamin E, 10% (wt%) DMSO were added to finally obtain control cryopreservation solution 2.
(2) Freezing and storing sperms: the control cryopreservation solution 2 and 10 groups of sperms are mixed uniformly in a cryopreservation tube according to the volume of 2:1, incubated on ice for 20 minutes, transferred to a place 4 cm above liquid nitrogen for staying for 15 minutes, and then the cryopreservation tube is immersed in liquid nitrogen for preservation.
(3) Thawing and recovering sperms: after the sperm are frozen for 11 months, the freezing tube filled with the sperm is taken out of a refrigerator at the temperature of-150 ℃, then immediately unfrozen in water bath at the temperature of 37 ℃ by rapid and gentle shaking, and immediately placed on ice (at the temperature of 0-4 ℃) for dark storage after unfreezing.
(4) And (3) sperm survival rate detection: the number of dead sperm stained with PI in reddish fluorescent color and all sperm stained with DAPI in bluish fluorescent color were counted separately by diluting thawed semen with control sperm base dilution solution 2, and observing with Propidium Iodide (PI) at 10. mu.g/ml and 4', 6-diamidino-2-phenylindole (DAPI) at 10. mu.g/ml under a fluorescence microscope to calculate the ratio of viable sperm. And secondly, diluting the thawed sperms by using a contrast sperm basic dilution solution 2, staining the sperms by using 1% trypan blue staining solution and activated sperms, and observing the sperms under a microscope to evaluate the survival rate of the sperms. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after 11 months of frozen storage in the control frozen storage solution 2, the survival rate of 10 groups of sparus latus (3823/4325, 88.4%) with specific results shown in table 1.
(5) And (3) sperm motility detection: and (3) taking the thawed semen, activating the sperm by using sterilized seawater with the same volume of 25 per mill, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm to total sperm. After 11 months of cryopreservation in the control cryopreservation solution 2, the motility of the sperm of 10 groups of yellow fin sea bream was (342/724, 47.2%), and the specific results are shown in table 1.
Comparative example 3
(1) Preparation of control cryopreservation solution 3: control sperm base dilution solution 3 was prepared by mixing Cortland solution and Hank's solution at a volume ratio of 1: 2. In control sperm cell base dilution solution 3, based thereon, trehalose 38.7g/L, sucrose 6.85g/L, 5% (wt%) BSA, 10% (wt%) DMSO were added, and control cryopreservation solution 3 was finally obtained.
(2) Freezing and storing sperms: the control cryopreservation solution 3 and 10 groups of sperms are mixed uniformly in a cryopreservation tube according to the volume of 2:1, incubated on ice for 20 minutes, transferred to a place 4 cm above liquid nitrogen for staying for 15 minutes, and then the cryopreservation tube is immersed in liquid nitrogen for preservation.
(3) Thawing and recovering sperms: after the sperm are frozen for 11 months, the freezing tube filled with the sperm is taken out of a refrigerator at the temperature of-150 ℃, then immediately unfrozen in water bath at the temperature of 37 ℃ by rapid and gentle shaking, and immediately placed on ice (at the temperature of 0-4 ℃) for dark storage after unfreezing.
(4) And (3) sperm survival rate detection: the number of dead sperm stained with PI in reddish fluorescent color and all sperm stained with DAPI in bluish fluorescent color were counted separately by diluting thawed semen with the control sperm base dilution solution 3, and observing with 10. mu.g/ml of Propidium Iodide (PI) and 10. mu.g/ml of 4', 6-diamidino-2-phenylindole (DAPI) under a fluorescence microscope, thereby calculating the proportion of viable sperm. And secondly, diluting the thawed sperms by using a contrast sperm basic dilution solution 3, staining the sperms by using 1% trypan blue staining solution and activated sperms, and observing the sperms under a microscope to evaluate the survival rate of the sperms. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after 11 months of frozen storage in the control frozen storage solution 2, the survival rate of 10 groups of sparus latus sperm is (4115/4769, 86.3%), and the specific results are shown in table 1.
(5) And (3) sperm motility detection: and (3) taking the thawed semen, activating the sperm by using sterilized seawater with the same volume of 25 per mill, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm to total sperm. After 11 months of cryopreservation in the control cryopreservation solution 3, the motility of the sperm of the red sea bream in 10 groups was (334/758, 44.1%), and the specific results are shown in table 1.
Comparative example 4
(1) Preparation of control cryopreservation solution 4: a control sperm cell base dilution solution 4 was prepared by mixing the Cortland solution and Hank's solution at a volume ratio of 1: 2. In control sperm cell base dilution solution 4, based thereon, trehalose 38.7g/L, sucrose 6.85g/L, 4% (wt%) BSA, 8. mu. mol/L vitamin E, 10% (wt%) DMSO were added to finally obtain control cryopreservation solution 4.
(2) Freezing and storing sperms: the control cryopreservation solution 4 and 10 groups of sperms were mixed in a cryopreservation tube at a volume of 2:1, incubated on ice for 20 minutes, transferred to a place 4 cm above liquid nitrogen for 15 minutes, and then the cryopreservation tube was immersed in liquid nitrogen for preservation.
(3) Thawing and recovering sperms: and after 11 months of cryopreservation, taking the cryopreservation tube filled with the sperms out of a refrigerator at-150 ℃, quickly and gently thawing in a water bath at 37 ℃ by shaking, and immediately placing on ice (0-4 ℃) for dark preservation after thawing.
(4) And (3) sperm survival rate detection: the number of dead sperm stained with PI in reddish fluorescent color and all sperm stained with DAPI in bluish fluorescent color were counted separately by diluting thawed semen with the control sperm base dilution solution 4, and observing with 10. mu.g/ml Propidium Iodide (PI) and 10. mu.g/ml 4', 6-diamidino-2-phenylindole (DAPI) under a fluorescence microscope to calculate the ratio of viable sperm. And secondly, diluting the thawed sperms by using a contrast sperm basic dilution solution 4, staining the sperms by using 1% trypan blue staining solution and activated sperms, and observing the sperms under a microscope to evaluate the survival rate of the sperms. After trypan blue staining and fluorescent observation, the survival rate of fresh sperms is 99.2%, and after the control cryopreservation solution 4 is frozen for 11 months, the survival rate of 10 groups of sparus latus sperms is (4557/5127, 88.9%), and the specific results are shown in table 1.
(5) And (3) sperm motility detection: and (3) taking the thawed semen, activating the sperm by using sterilized seawater with the same volume of 25 per mill, taking a small amount of sperm solution, observing the sperm under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm. And (3) vitality evaluation: the percentage of linearly rapidly moving sperm to total sperm. After 11 months of cryopreservation in the control cryopreservation solution 2, the motility of the sperm of 10 groups of yellow fin sea bream was (392/816, 48.1%), and the specific results are shown in table 1.
TABLE 1 statistics of post-resuscitation sperm survival (%) and motility (%) in different examples and comparative examples
As can be seen from table 1, in examples 1 to 4, the average survival rates of the rhabdosargus sargus sperms after being frozen and stored for 11 months are all higher than 90%, and the average activities are not lower than 49%, which indicates that the rhabdosargus sargus sperms frozen and stored solution ensures the survival rate and the activity of the rhabdosargus sargus sperms after being frozen and stored; and when the addition amount of the melatonin is 8-10 mu mol/L, the survival rate and the vitality of the sperms of the yellow fin porgy after being frozen are the best, and the optimal effect cannot be achieved when the proportion of the melatonin is too low or too high.
Comparing the example 1 with the comparative example 1, it can be seen that the melatonin and the vitamin E are added into the cryopreservation liquid of the sperms of the oplegnathus fasciatus, so that the average survival rate and the average vitality of the sperms of the oplegnathus fasciatus after cryopreservation are improved by more than 10 percent; comparing example 1 with comparative example 2, it can be seen that melatonin is added into the cryopreservation solution for the sperm of the yellow fin sea bream, so that the average survival rate and the average vitality of the sperm of the yellow fin sea bream after cryopreservation are obviously improved, wherein the average vitality is improved by more than 10%.
As can be seen from comparative example 3, when melatonin and vitamin E were not added to the cryopreservation solution, even if the amount of BSA added was increased by two times, the average survival rate and average motility of the yellow fin sea bream sperm were still much lower than those of example 1; from comparative example 4, it is known that the average survival rate and average motility of the sperm of the oplegnathus fasciatus are still lower than those of examples 1 to 4 when melatonin is not added to the cryopreservation liquid but a large amount of BSA and vitamin E are added. The above results demonstrate that the antioxidant combination of bovine serum albumin, melatonin and vitamin E of the present invention is used in the sparus latus sperm cryopreservation solution, and improves the survival rate and viability of sparus latus sperm after cryopreservation.
Example 5
Mixing experiment of cortland solution and Hank's solution in different proportions
Sperm base dilutions were set at different cortland and Hank's solutions mixing ratios (mixed by volume):
control solution 1: a 100% corrland solution of a water-soluble polymer,
control solution 2: a 100% solution of Hank's,
test solution 1: the mortland solution and the Hank's solution were mixed at 3:1,
test solution 2: the mortland solution and the Hank's solution were mixed at a ratio of 2:1,
test solution 3: the mortland solution and the Hank's solution were mixed at a ratio of 1:1,
test solution 4: the mortland solution and Hank's solution were mixed at a ratio of 1:2,
test solution 5: the mortland solution and the Hank's solution were mixed at 1:3,
test solution 6: the mortland solution and Hank's solution were mixed at a ratio of 1: 4.
The freshly collected sperms are respectively diluted by 10 times by the different sperm basic diluents and divided into 3 groups, each group is activated by sterilized seawater with the same volume of 25 per thousand, and then observed under an inverted microscope with the volume of 400 times, and the proportion of the sperms in linear motion, namely the activity of the sperms is counted, and the result is shown in table 2.
As can be seen from Table 2, compared with a single corrland solution or Hank's solution, namely comparative solutions 1 and 2, the sperm motility of the yellow fin sea bream after being diluted by experimental solutions 2-5 is obviously improved. Namely, the cortland solution and the Hank's solution are mixed according to the mixing volume ratio of 2: 1-1: 3 to be used as a sperm base diluent, so that the sperm motility of the yellow fin sea bream can be obviously improved.
TABLE 2 statistics of sperm motility (%) after sperm dilution and activation with different sperm base dilutions
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments of the present invention are to be considered in all respects as illustrative and not restrictive. Therefore, any minor modifications, equivalent changes and modifications to the above embodiments according to the spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (6)
1. A cryopreservation solution for sparus latus sperms is characterized in that: comprises sperm basic diluent, nutrient substances, an antioxidant and an anti-freezing protective agent; the sperm base diluent is formed by mixing a Cortland solution and a Hank's solution according to the volume ratio of 2: 1-1: 3; the nutrient substances comprise sucrose and trehalose; the antioxidant comprises bovine serum albumin, melatonin and vitamin E; the anti-freezing protective agent is dimethyl sulfoxide;
wherein, the sperm basic diluent is taken as a reference, the addition amount of bovine serum albumin in percentage by mass is 1-3%, the addition amount of melatonin is 1-100 mu mol/L, and the addition amount of vitamin E is 1-10 mu mol/L; the addition amount of trehalose is 35.0-45.0 g/L, the addition amount of sucrose is 6-7 g/L, and the addition amount of dimethyl sulfoxide is 5-15% by mass.
2. The cryopreservation solution for sperm of oplegnathus fasciatus as claimed in claim 1, wherein: the volume ratio of Cortland solution to Hank's solution is 1: 2.
3. The cryopreservation solution for yellow fin sea bream sperm according to claim 1 or 2, wherein: the addition amount of bovine serum albumin is 2 percent by mass, the addition amount of melatonin is 10 mu mol/L, and the addition amount of vitamin E is 5 mu mol/L.
4. The cryopreservation solution for sperm of yellow fin sea bream according to claim 3, wherein: the addition amounts of trehalose and sucrose are respectively 38.7g/L and 6.85 g/L; the addition amount of the dimethyl sulfoxide is 10 percent by mass.
5. A cryopreservation method of the sperm of the Sparus latus using the cryopreservation solution as defined in any one of claims 1 to 4, comprising the steps of:
s1, semen collection: in the breeding season of the sparus latus, selecting healthy male parent fish with mature spermary, wiping water around a cloaca of a fish body with a clean towel, lightly pressing the abdomen until the cloaca flows out milky semen, and collecting the semen with a centrifugal tube;
s2, preparing the sparus latus sperm cryopreservation solution;
s3, freezing and storing sperm: uniformly mixing the sparus latus sperm cryopreservation solution and the semen in a cryopreservation tube, and diluting the semen; firstly, placing a freezing tube on ice, incubating and keeping the freezing tube away from light for 10-30 minutes, then placing the tube above the surface of liquid nitrogen for freezing and staying for 10-30 minutes, then immersing the tube in the liquid nitrogen for rapid freezing treatment, and finally transferring the tube to a low temperature of-150 ℃ for preservation;
s4, sperm recovery: after the sperm are frozen for a period of time, taking out the cryopreservation tube filled with the sperm from liquid nitrogen, then immediately and rapidly thawing in a 37 ℃ water bath, and preserving the thawed sperm at 0-4 ℃;
s5, sperm survival rate detection: diluting the thawed sperms with a sperm basic diluent, dyeing the sperms with trypan blue staining solution, and observing under a microscope to evaluate the survival rate of the sperms; secondly, diluting the thawed sperms by using a sperm basic dilution solution, and observing by using propidium iodide and 4', 6-diamidino-2-phenylindole under a fluorescence microscope to count the survival rate of the sperms;
s6, sperm motility detection: and taking the thawed semen, activating the sperm, taking a small amount of sperm solution, observing under a 400-time inverted microscope, and evaluating the sperm motility according to the movement state of the sperm solution.
6. Use of the cryopreservation solution of any one of claims 1 to 4 in cryopreservation of sperm of oplegnathus fasciatus.
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Non-Patent Citations (3)
| Title |
|---|
| Antioxidants in Fish Sperm and the Potential Role of Melatonin;Francisca Félix 等;《Antioxidants》;20201203;第10卷(第36期);第1-29页 * |
| Effects of melatonin supplementation on the quality of cryopreserved sperm in the neotropical fish Prochilodus lineatus;Naiara Cristina Motta 等;《Theriogenology》;20211101;第14-21页 * |
| 鱼类精液保存技术研究进展前景展望;陈健等;《渔业致富指南》;20180425;第22-24页 * |
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