CN107047540B - Ultralow-temperature preservation method for seminal fluid of leiocassis longirostris and diluent thereof - Google Patents
Ultralow-temperature preservation method for seminal fluid of leiocassis longirostris and diluent thereof Download PDFInfo
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Abstract
The invention discloses a preservation diluent and an ultralow temperature preservation method for leiocassis longirostris seminal fluid. The preservation diluent consists of sugar, sodium salt, potassium salt, calcium salt, tyrosine, glycine, distilled water and an antifreeze agent dimethyl sulfoxide. The ultra-low temperature preservation method comprises the following steps: selecting excellent male parent fish meeting the conditions for semen collection, storing in a preservation diluent added with neomycin sulfate and streptomycin, adopting a cooling rate of-10.5 ℃/30s, and after the temperature reaches-150 ℃, placing in liquid nitrogen for ultralow-temperature preservation; when thawing recovery is needed, taking out the leiocassis longirostris semen preserved at ultralow temperature, adding PBS buffer solution with the volume fraction of 0.65-0.7% as thawing solution, and quickly thawing and recovering in a water bath at the temperature of 32-40 ℃. The technology can ensure the durability of the leiocassis longirostris semen preservation time, has good protection effect on the semen, and has high survival rate and good activity of the preserved semen.
Description
Technical Field
The invention belongs to the technical field of aquaculture. More particularly relates to a semen ultralow-temperature preservation method of rare and easy-to-risk fish leiocassis longirostris and a diluent thereof.
Background
Leiocassis longissima is of Leiocassis of Clariales of Claria. Body length, lateral flattening, dorsal fin starting point as the highest point of the body. The head is flat and slightly triangular, and the back bone is rough and exposed. The anastomoses are prominent and the circle is blunt. The proximal part of the mouth is arc-shaped, and the upper jaw is slightly protruded. Is fish at the bottom of river and stream of subtropical hills, which is fond of a clear running water environment. The leiocassis longirostris has no muscle stings, delicious meat taste and high nutritional value, so the leiocassis longirostris is high in price in the market and the condition of short supply and demand often occurs. Wild leiocassis longirostris is caught excessively, so that wild resources and young fishes of the leiocassis longirostris are deficient (wild resources such as seedlings and the like are deficient). Meanwhile, leiocassis longissima is one of the most famous and high-quality economic fishes in the river basin and with the greatest difficulty in artificial breeding. Even though some domestic scholars are dedicated to the artificial reproduction of leiocassis longirostris and the basic research of fertilization biology for a long time, the key factors influencing the survival rate of the artificial fertilization offspring seed of leiocassis longirostris are sought, but the effect is very little.
On the basis of the successful initial artificial propagation of leiocassis longirostris in the Yangtze river, the subject group discovers that the main factors for restricting the artificial propagation of the leiocassis longirostris are not traditional environmental ecological factors such as water temperature and pH and are not good in egg quality of female parent fishes, the problem can be caused on male parent fishes and is related to the gonad development state of the male parent fishes, the male parent fishes with good development are difficult to obtain, even if the male parent fishes with good development are occasionally obtained, the female parent fishes can not develop and mature, and the phenomenon that the female and male fishes develop asynchronously is the bottleneck problem that the artificial propagation of the leiocassis longirostris cannot be successful. Therefore, the preservation of the seminal fluid of the male leiocassis longirostris is very important, and related researches and reports are not found at present.
Disclosure of Invention
The invention aims to overcome the defects of the existing leiocassis longirostris semen preservation technology, provides a leiocassis longirostris semen dilution liquid and an ultralow temperature preservation method, provides guarantee for germplasm research and genetic management, semen cryopreservation, germplasm protection and the like, and provides important scientific guidance for solving the technical problem of artificial propagation of leiocassis longirostris, protecting leiocassis longirostris resources and realizing healthy and sustainable development of famous and high-quality fish breeding industry in China.
The invention aims to provide a leiocassis longirostris semen preservation diluent.
The invention also aims to provide a leiocassis longirostris seminal fluid ultralow-temperature preservation method.
The above purpose of the invention is realized by the following technical scheme:
a dilution for preserving the semen of Leiocassis longissima comprises sugar, sodium salt, potassium salt, calcium salt, tyrosine, glycine, distilled water and antifreeze; the antifreeze agent is dimethyl sulfoxide.
Wherein, preferably, the sugar is glucose, or the sugar is glucose and fructose.
Preferably, the sodium salt is NaCl, NaHCO3Or Na2HPO4One or more of them.
Preferably, the potassium salt is KCl, or the potassium salt is KCl and KH2PO4。
Preferably, the calcium salt is CaCl2。
Preferably, the leiocassis longirostris semen preservation diluent also comprises magnesium salt; preferably, the magnesium salt is MgCl2Or MgSO 24。
The neomycin sulfate and streptomycin are added into the leiocassis longirostris semen preservation diluent before use. Preferably, the addition volume ratio of the neomycin sulfate to the streptomycin is 1-1.2: 1.
particularly preferably, the leiocassis longirostris semen preservation diluent disclosed by the invention has the following three formulas of A, B, C:
a formula: each 100mL of distilled water contains 0.15-0.2 g of glucose, 1.5-1.6 g of NaCl, 0.15-0.18 g of KCl, and 0.02-0.03 g of CaCl2、0.04～0.05g MgSO4·7H2O、0.06～0.07g NaHCO3、0.03～0.04g Na2HPO4·H2O、0.01～0.015g KH2PO4、0.18～0.2g C6H12O6·H2O, dimethyl sulfoxide with the final volume concentration of 5-15%;
and B, formulation: each 100mL of distilled water contains 1.5-2 g of glucose, 1-1.5 g of fructose, 0.05-0.1 g of KCl, 0.01-0.03 g of CaCl2、0.18～0.25g NaHCO3Dimethyl sulfoxide with the final volume concentration of 5-15%;
and C, formulation: each 100mL of distilled water contains 2.5-3 g of glucose, 0.8-1 g of NaCl, 0.1-0.2 g of KCl, and 0.02-0.04 g of CaCl2、0.01～0.02g MgCl2·6H2O、0.05～0.08g Na2HPO4、0.3～0.4g NaHCO30.1 to 0.2g of tyrosine, 0.05 to 0.1g of glycine and dimethyl sulfoxide with the final volume concentration of 5 to 15 percent.
More preferably, the A, B, C formula of the leiocassis longirostris semen preservation diluent is as follows:
a formula: each 100mL of distilled water contains 0.18g of glucose, 1.52g of NaCl, 0.168g of KCl, and 0.028g of CaCl2、0.0472g MgSO4·7H2O、0.065g NaHCO3、0.035g Na2HPO4·H2O、0.012g KH2PO4、0.188g C6H12O6·H2O, dimethyl sulfoxide with a final volume concentration of 10%;
and B, formulation: each 100mL of distilled water contains 1.8g of glucose, 1.2g of fructose, 0.08g of KCl and 0.02g of CaCl2、0.21g NaHCO3Dimethyl sulfoxide at a final volume concentration of 10%;
and C, formulation: each 100mL of distilled water contains 2.68g of glucose, 0.98g of NaCl, 0.16g of KCl and 0.03g of CaCl2、0.012g MgCl2·6H2O、0.06g Na2HPO4、0.32g NaHCO30.12g tyrosine, 0.08g glycine, 10% final volume concentration of dimethyl sulfoxide.
The sperm preservation solution is preserved at low temperature by using a sealable brown glass bottle for later use.
Of the three semen preserving diluent formulas, the formula C has the most obvious effect.
In addition, the ultralow temperature preservation method of the seminal fluid of the leiocassis longirostris comprises the following steps:
s1, selecting fish species: selecting male parent fish with no disease and obvious sexual maturity characteristics;
s2, preparing a semen preservation diluent;
s3, semen collection: semen collection is carried out on the male parent fish in the step S1;
s4, semen ultralow-temperature preservation: adding neomycin sulfate and streptomycin into the semen preservation diluent in the step S2, and mixing the obtained mixture according to the ratio of semen: stock dilution = 1: 5-6, storing the semen collected in S3 in a storage diluent; then, the temperature is reduced at a speed of-10 to-11 ℃/30s, and the mixture is put into liquid nitrogen for ultralow temperature preservation after reaching-150 ℃;
s5, thawing recovery: taking out the leiocassis longirostris semen preserved at ultralow temperature, adding the unfreezing liquid, and quickly unfreezing and recovering in a water bath at the temperature of 32-40 ℃; the thawing solution is PBS buffer solution with the volume fraction of 0.65-0.7%.
Preferably, the fish species selection in step S1 is specifically: firstly, selecting and culturing an individual with no damage to the appearance, large age (mature period) and heavy individual (more than 3 jin) for intensive culture; then selecting male parent fish with strong and healthy constitution, no damage to body surface, no congestion, abdominal distention, fin rot, exophthalmos and other diseases, obvious sexual maturity characteristic, strong constitution, correct shape, stable swimming posture and bright body color to collect semen. This method is extremely important for later semen preservation.
Preferably, the specific method for collecting semen in step S3 can be referred to as follows: wiping the fish body to remove urine and excrement before semen collection, narcotizing parent fish with MS-222 anesthetic, quickly taking out the spermary from an aseptic operation table, weighing, completely shearing the spermary with scissors to fully release sperms, and collecting sperm suspension. The whole process is carried out on ice, and the sperm suspension comprises sperms, spermatids, seminal plasma, blood cells, micro tissue fragments and the like.
Preferably, step S4 the neomycin sulfate or streptomycin: the volume ratio of the stored diluent is 1-1.2: 1.
preferably, the cooling rate of step S4 is-10.5 ℃/30S.
Preferably, the container used in step S4 is a plastic centrifuge tube, that is, the semen preservation diluent in step S2 is added into the plastic centrifuge tube, and neomycin sulfate and streptomycin are added, according to the ratio of semen: stock dilution = 1: 5-6, adding the semen collected in S3 into a plastic centrifuge tube; then, the plastic centrifuge tube is directly immersed into liquid nitrogen for ultralow temperature preservation after the temperature reaches-150 ℃ by adopting the descending speed of-10.5 ℃/30 s.
Preferably, the thawing recovery temperature of step S5 is 36 ℃.
The invention has the following beneficial effects:
the leiocassis longirostris semen diluent and the ultralow temperature preservation and recovery method provided by the invention can ensure the duration of the leiocassis longirostris semen preservation time, and the semen ultralow temperature preservation technology greatly promotes the development and breeding improvement of leiocassis longirostris germplasm resources, and has important theoretical and practical significance in leiocassis longirostris breeding. Firstly, the original fine breed of the good leiocassis longirostris can be preserved for a long time by establishing a frozen sperm bank for the good breeding of the leiocassis longirostris, so that the phenomena of germ plasm resource degradation and variation caused by killing of leiocassis longirostris species or production long-term breeding and inbreeding due to transitional fishing, environmental pollution and ecological damage are avoided; and secondly, through the ultralow temperature preservation of sperms, bred strains which are asynchronous, sex-reversed and geographically isolated in gonad development and reproductive period can be mated, germplasm resources and natural preservation quantity are recovered, further industrial and large-scale production can be realized, and considerable ecological economic and social benefits are achieved.
Specifically, the technical scheme of the invention has the following remarkable innovations and significances:
1. the improved sampling method comprises the following steps: the rare and dangerous leiocassis longirostris is used as an object of the invention, wild leiocassis longirostris parent fishes of different geographic populations are collected, and the problem of asynchronous male and female gonad development is primarily solved by using a semen ultralow temperature preservation technology.
2. The formula of the improved preservation diluent comprises the following steps: according to the biological and gonad characteristics of the leiocassis longirostris and the survival rate of sperms after the leiocassis longirostris is stored by different diluent formulas, the leiocassis longirostris sperm storage diluent formula is further improved, 3 excellent formulas are selected, the effect is good, and the effect is obvious particularly by the formula C.
3. Improving the semen protective agent: the sperm protective agent adopts neomycin sulfate and streptomycin complexing agent in a certain proportion to replace pure penicillin, and has good protection effect on sperm. The concentration of the sperm antifreeze agent is groped, and the addition of 10 percent of dimethyl sulfoxide has the best effect on the sperm preserved at ultralow temperature.
4. Optimizing a cooling method: the ultra-low temperature preservation uses a step-by-step rapid cooling method, is greatly improved on the basis of the prior people, and has positive significance on the preservation effect of sperms.
5. Improving frozen semen thawing solution: the leiocassis longirostris frozen semen thawing solution uses PBS buffer solution with the volume fraction of 0.65-0.7% to replace normal saline or distilled water, and has better protection effect on semen.
Drawings
FIG. 1 is a diagram showing the results of Percoll separation of semen.
FIG. 2 shows the results of a 3-layer Percoll discontinuous gradient separation of semen.
Fig. 3 shows the sperm motility rate and survival rate of leiocassis longirostris after the sperm is frozen and preserved for 30 days at ultralow temperature.
FIG. 4 shows the effect of different resuscitation temperatures on sperm motility.
FIG. 5 shows the recovered sperm observed under a microscope at a magnification of 16X 10.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Leiocassis longissima semen preservation diluent
1. Sperm preservation solution dilution A formula: respectively preparing liquid A and liquid B, and optimally preparing the liquid A according to the grope and the experimentThe method comprises the following steps: glucose 1.80g, NaCl 15.2g, KCl 1.68g, CaCl2 0.28g，MgSO4·7H20.472g of O, and finally adding 100ml of distilled water for dissolving;
the formula of the solution B is NaHCO3 0.65g，Na2HPO4·H2O 0.35g，KH2PO4 0.12g，C6H12O6·H2O1.88 g, and finally 100ml of distilled water is added for dissolution.
Mixing the first solution and the second solution 10mL respectively, adding distilled water to constant volume of 100mL, and adding dimethyl sulfoxide with final volume concentration of 5%, 10% or 15% to obtain sperm preserving diluent A.
2. The formula of the sperm preservation diluent B is as follows: 1.80g of glucose, 1.2g of fructose, 0.08g of KCl and CaCl2 0.02g，NaHCO30.21g, 100mL of distilled water, and dimethyl sulfoxide at a final concentration of 5%, 10% or 15% by volume.
3. The formula of the sperm preservation diluent C is as follows: 2.68g of glucose, 0.98g of NaCl, 0.16g of KCl and CaCl20.03g，MgCl2·6H2O 0.012g，Na2HPO4 0.06g，NaHCO30.32g tyrosine 0.12g glycine 0.08g distilled water 100mL, and adding final volume concentration of 5%, 10% or 15% dimethyl sulfoxide.
Tests prove that the survival rate of sperms after the 3 excellent formulas are stored is better, and particularly the effect of the formula C is most obvious. The volume concentration of dimethyl sulfoxide is preferably 10%.
Example 2 Leiocassis longissima semen ultra-low temperature preservation method
The invention provides a method for preserving the semen of leiocassis longirostris at ultralow temperature, which mainly comprises the following steps: selecting fish species, preparing a sperm preservation diluent and an antifreeze agent, collecting and observing sperm quality, preserving sperm at ultralow temperature, thawing and reviving, and observing sperm motility. The specific operation method is as follows:
1. fingerling selection
The fish species collection in the natural environment is an important parent fish source of the leiocassis longirostris, 300 tails of the leiocassis longirostris parent fish are introduced and collected through long-term field investigation, more than 100 tails of individuals with no appearance damage, larger age and heavier weight of 3 jin are bred, and the intensive cultivation is carried out in scientific research demonstration bases of Xingxing fishery and aquaculture science and technology Limited in Qingyuan. When collecting semen, selecting male parent fish with strong constitution, no damage to body surface, no congestion, abdominal distention, fin rot, exophthalmos, etc., and has the advantages of obvious sexual maturity, strong constitution, correct shape, stable swimming posture, and bright body color. This method is extremely important for later semen preservation.
2. Preparation of sperm preservation diluent
Sperm cell preservation dilutions were prepared according to example 1.
3. Sperm collection, separation and quality observation
(1) Sperm collection chooses for use 10mL can seal light-resistant centrifuging tube, and as sperm preservation container after sterilization process, totally 60, every kind of preservative solution 20 will preserve in the liquid careful addition centrifuging tube to respectively according to 1 ~ 1.2: neomycin sulfate and streptomycin are added according to the proportion of 1, and neomycin sulfate and streptomycin are not added in a control group and numbered.
Wiping the fish body to remove urine and excrement before semen collection, narcotizing parent fish with MS-222 anesthetic, quickly taking out the spermary on a sterile operating table, weighing, and completely shearing the spermary with scissors to release sperms. The collected sperm suspensions were filtered through a gauze and labeled.
The whole process is carried out on ice, and the sperm suspension comprises sperms, spermatids, seminal plasma, blood cells, micro tissue fragments and the like. Isotonic 100% Percoll solution Percoll (Pharmacia, Uppsala, Sweden) stock solution and 10 XPBS 9: 1, and mixing the components uniformly. Subsequent Percoll gradients were diluted (10% to 90%) with 1 XPBS as required for the experiment, using three size sterile centrifuge tubes.
(2) Performing microscopic examination after the collection is finished, taking 2 microliter semen to observe under a microscope, wherein the activity of the semen is as follows: about 82% of the sperm cells can move forward quickly and linearly, 9% of the sperm cells can move or swing slightly in a curve, 6% of the sperm cells rotate in situ, 3% of the sperm cells are still, and the sperm cell survival rate is 0.82.
Further using a computer sperm auxiliary analyzer to detect and display that the pH value of the fresh semen is 7.1.
The separation result of leiocassis longirostris sperm Percoll is shown in the attached figure 1, and the separation result of 3-layer Percoll discontinuous gradient is shown in the attached figure 2.
4. Cryopreservation of semen
(1) The sperm were split into two parts, one for cryopreservation experiments and the other half for cryopreservation experiments. The freezing method adopts a plastic centrifuge tube freezing method, has the characteristics of large freezing quantity, simple and convenient operation, high safety and the like, and can be used for semen freezing preservation and semen bank establishment.
Taking out semen, measuring semen volume, placing in a centrifuge tube with equal volume, wherein the ratio of semen to preservation solution is 1: 5-6, the method is simple and convenient, low in cost and suitable for popularization and application in production.
(2) Specifically, we performed cryopreservation experiments on sperm, and the results of cryopreservation of sperm at 4 ℃ are shown in table 1 below:
TABLE 1 semen preservation at 4 deg.C
Note: "+ +" indicates that more than 80-90% of the sperm move rapidly; "+" indicates that 50-60% of the sperm move rapidly; "+ -" indicates that 20-30% of the sperm move rapidly; "-" indicates total death or a small amount of vibration in place.
We have found that cooling rates that are either too fast or too slow can cause cell damage and reduce the survival rate of leiocassis longirostris sperm. Therefore, the collected semen is preserved at low temperature by adopting a step-by-step rapid cooling method, the specific method is to adopt a decreasing rate of-10.5 ℃/30s for the sperms of the leiocassis longirostris, and the sperms are directly immersed in liquid nitrogen after reaching the low temperature of-150 ℃, so that the method has good effect and can protect the sperms to the maximum extent, and each sperm preservation solution is preserved and observed in 3 groups. The specific method is that the leiocassis longirostris sperms are taken out and thawed after being frozen at ultralow temperature for 30 days, and after thawing, the influence of adding 5% of dimethyl sulfoxide and 10% of dimethyl sulfoxide on the sperm motility rate of frozen and preserved is not remarkably different (37.6% and 36.2%) but is remarkably higher than 15% of dimethyl sulfoxide group (24.4%), but the 10% of dimethyl sulfoxide keeps 46.6% and is remarkably higher than 37.2% of 5% of dimethyl sulfoxide group in terms of sperm survival rate (as shown in figure 3). In summary, the addition of 10% dimethyl sulfoxide works best as an antifreeze.
5. Thawing recovery
Thawing recovery is a sperm warming, dissolving and recovering process and is also a sperm vitality recovering process, and the key factor of thawing is the thawing rate and the thawing rate.
Repeated tests and groping prove that the leiocassis longirostris frozen sperm thawing solution has a good effect by using a PBS (phosphate buffer solution) with the volume fraction of 0.65-0.7%.
The thawing speed adopts a quick thawing method, and the specific operation is quick thawing in a water bath kettle at the temperature of 32-40 ℃. The method corresponds to the process of quick freezing and has good effect.
The influence of different recovery temperatures on the activity of the thawed leiocassis longirostris sperms is comparatively analyzed, and the activity of the recovered leiocassis longirostris sperms is the highest and is 30.66% when the recovery temperature is 36 ℃. The effect of specific different resuscitation temperatures on sperm motility is shown in figure 4.
6. Observation of sperm motility
The preservation effect evaluation was performed by using the activated sperm motility (percentage of fast moving sperm to the total sperm count) as an index. Taking out the sperm preservation solution, shaking uniformly, quickly sucking out a small drop of the sperm preservation solution by using a pipette, placing the small drop of the sperm preservation solution on a glass slide, observing the sperm under the condition of a microscope with the magnification of 16 multiplied by 10, immediately activating the observed sperm by using a half drop of distilled water, observing the movement condition of the observed sperm and recording the movement condition. We found that the percentage of sperm moving forward in a straight line in the visual field was 45% or more, that of sperm moving in a flutter, a wobble or a faint curve in situ was 35% or so, and that the number of sperm which were completely immobile was less than 20%, and that the sperm had no fertilization ability (the sperm was observed under a microscope at a magnification of 16X 10. fig. 5).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (5)
1. An ultralow-temperature preservation method for seminal fluid of leiocassis longirostris is characterized by comprising the following steps:
s1, selecting fish species: selecting male parent fish with no disease and obvious sexual maturity characteristics;
s2, preparing a semen preservation diluent;
s3, semen collection: semen collection is carried out on the male parent fish in the step S1;
s4, semen ultralow-temperature preservation: adding neomycin sulfate and streptomycin into the semen preservation diluent in the step S2, and mixing the obtained mixture according to the ratio of semen: the stock dilutions were 1: 5-6, storing the semen collected in S3 in a diluent; then, adopting a cooling rate of-10.5 ℃/30s, and after the temperature reaches-150 ℃, putting the mixture into liquid nitrogen for ultralow temperature preservation;
s5, thawing recovery: taking out the leiocassis longirostris semen preserved at ultralow temperature, adding the unfreezing liquid, and quickly unfreezing and recovering in a water bath at the temperature of 32-40 ℃; the thawing solution is PBS buffer solution with the volume fraction of 0.65-0.7%;
the formula of the semen preservation diluent in the step S2 is as follows: each 100mL of distilled water contains 0.15-0.2 g of glucose, 1.5-1.6 g of NaCl, 0.15-0.18 g of KCl, and 0.02-0.03 g of CaCl2、0.04～0.05g MgSO4·7H2O、0.06～0.07g NaHCO3、0.03～0.04g Na2HPO4·H2O、0.01～0.015g KH2PO4、0.18～0.2g C6H12O6·H2O, dimethyl sulfoxide with a final volume concentration of 10%;
or the formula is as follows: each 100mL of distilled water contains 1.5-2 g of glucose, 1-1.5 g of fructose, 0.05-0.1 g of KCl, 0.01-0.03 g of CaCl2、0.18～0.25g NaHCO3Dimethyl sulfoxide at a final volume concentration of 10%;
or the formula is as follows: each 100mL of distilled water contains 2.5-3 g of glucose, 0.8-1 g of NaCl, 0.1-0.2 g of KCl, and 0.02g of～0.04g CaCl2、0.01～0.02g MgCl2·6H2O、0.05～0.08g Na2HPO4、0.3～0.4g NaHCO30.1 to 0.2g of tyrosine, 0.05 to 0.1g of glycine, and dimethyl sulfoxide with a final volume concentration of 10%.
2. The method of claim 1, wherein the semen preserving diluent is formulated as: each 100mL of distilled water contains 0.18g of glucose, 1.52g of NaCl, 0.168g of KCl, and 0.028g of CaCl2、0.0472g MgSO4·7H2O、0.065g NaHCO3、0.035g Na2HPO4·H2O、0.012g KH2PO4、0.188g C6H12O6·H2O, dimethyl sulfoxide with a final volume concentration of 10%;
or the formula is as follows: each 100mL of distilled water contains 1.8g of glucose, 1.2g of fructose, 0.08g of KCl and 0.02g of CaCl2、0.21g NaHCO3Dimethyl sulfoxide at a final volume concentration of 10%;
or the formula is as follows: each 100mL of distilled water contains 2.68g of glucose, 0.98g of NaCl, 0.16g of KCl and 0.03g of CaCl2、0.012g MgCl2·6H2O、0.06g Na2HPO4、0.32g NaHCO30.12g tyrosine, 0.08g glycine, 10% final volume concentration of dimethyl sulfoxide.
3. The method according to claim 1, wherein the fish species selection in step S1 is specifically: firstly, selecting and breeding individuals with no injury and disease in appearance, mature period and weight not less than 3 jin for intensive culture; then selecting male parent fish with strong and healthy constitution, no damage to body surface, no congestion, abdominal distention, fin rot, and sudden eye disease, and with obvious sexual maturity characteristic, strong constitution, correct shape, stable swimming posture, and bright body color to collect semen.
4. The method of claim 1, wherein step S4 is performed on the neomycin sulfate or streptomycin: preserving the volume ratio of the diluent to 1-1.2: 1.
5. the method of claim 1, wherein the thawing recovery temperature of step S5 is 36 ℃.
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| CN1596670A (en) * | 2004-07-27 | 2005-03-23 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
| CN103348966A (en) * | 2013-05-31 | 2013-10-16 | 中国科学院海洋研究所 | Method for efficient ultralow temperature cryopreservation of turbot sperms |
| CN105052893A (en) * | 2015-07-29 | 2015-11-18 | 上海市水产研究所 | Ultralow temperature cryopreservation method of collichthys lucidus sperm |
| CN105494314A (en) * | 2015-12-17 | 2016-04-20 | 武汉先锋水产科技有限公司 | Ancherythroculter nigrocauda sperm preservation method |
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