CN106417256B - method for improving sperm utilization rate of rainbow trout pseudo-male fish - Google Patents

method for improving sperm utilization rate of rainbow trout pseudo-male fish Download PDF

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CN106417256B
CN106417256B CN201610891101.1A CN201610891101A CN106417256B CN 106417256 B CN106417256 B CN 106417256B CN 201610891101 A CN201610891101 A CN 201610891101A CN 106417256 B CN106417256 B CN 106417256B
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pseudo
male fish
rainbow trout
semen
fish
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CN106417256A (en
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谷伟
王淑梅
王炳谦
户国
徐革锋
孙鹏
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Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
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Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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Abstract

method for improving the sperm utilization rate of rainbow trout pseudo-male fish, which relates to methods for improving the sperm utilization rate of the rainbow trout, aims to solve the problems of low sperm activation rate and low fish egg fertilization rate and eye growth rate of the rainbow trout pseudo-male fish processed by the existing methods, and comprises , preparing a sperm preservation solution of the rainbow trout pseudo-male fish, adjusting the pH value after the sperm preservation solution is prepared, placing the solution at 8-12 ℃ for standby, selecting high-quality and healthy rainbow trout pseudo-male fish, dissecting to obtain a pseudo-male fish spermary, placing the pseudo-male fish spermary in a clean and anhydrous mortar, grinding the mortar to obtain a sperm tissue mixed solution, filtering the mixed solution, mixing the pseudo-male fish spermary with the sperm preservation solution, placing the mixture in an oxygen bag, storing the mixture, taking out the mixture and then using the mixture for fish egg fertilization.

Description

method for improving sperm utilization rate of rainbow trout pseudo-male fish
Technical Field
The invention relates to methods for improving the utilization rate of sperms of pseudo-male fish.
Background
The rainbow trout has been developed for centuries, is of the earliest salmon trout breed, can effectively reduce the production cost by genetic improvement, adopts chromosome operation technology to cultivate polyploids, produces full-female diploid and triploid oosperm on a large scale, is of the important means, and the full-female triploid has the advantages that the growth advantages and the disease resistance caused by the long service life and the sterility in the maturation period are incomparable with diploid fish, is subject to the pursuit of a plurality of breeding units, imports a large amount of eyelets from abroad every year, and becomes the main breed of the rainbow trout at present.
The pseudo-male fish is a key technology for preparing the full-female triploid, the full-female triploid can be obtained by mating with a tetraploid female fish, or the full-female triploid can be obtained by hydrostatic pressure or temperature treatment of fertilized eggs obtained after mating with a diploid female fish, the pseudo-male fish is obtained by feeding 17 α methyltestosterone feed to a floating young fish individual to induce the reversion of a hereditary female phenotype into a male, however, the spermary of the pseudo-male fish is malformed and needs to obtain semen artificially, the sperms of the pseudo-male fish have no motility and the fertility rate is extremely low, in order to breed offspring by using the pseudo-male fish, the spermary semen of the pseudo-male fish needs to be cultured by a sperm preserving solution to obtain the vitality so as to carry out insemination activities, and in order to improve the sperm vitality of the pseudo-male fish and improve the fertility rate of fish eggs, the existing research results show that the fertility rate can reach 52% by improving the pH value of the artificial seminal plasma, and when the full-female triploid is produced, a great amount of fish eggs still needs to be wasted according to the existing method, namely, and the vitality of the sperm.
Disclosure of Invention
The invention provides methods for improving the sperm utilization rate of rainbow trout pseudo-male fish, aiming at solving the problems of low sperm activation rate and low fish egg fertilization rate and eye growing rate after the sperm of the rainbow trout pseudo-male fish is processed by the existing method.
The method for improving the sperm utilization rate of the rainbow trout pseudo-male fish comprises the following steps:
, preparing a seminal fluid preserving fluid for the rainbow trout pseudo-male fish, wherein the seminal fluid preserving fluid comprises 6-8 g/L NaCl, 2-4 g/L KCl and 0.2-0.4 g/L CaCl2·2H2O、0.2～0.4g/L MgCl2·6H2O、0.1～0.3g/L NaHCO3、4～5g/LC6H5Na3O7And distilled water.
Secondly, after the semen preserving fluid is prepared, the pH value is adjusted to 9-10, and then the semen preserving fluid is placed for 2-3 hours at the temperature of 8-12 ℃ for standby.
Selecting high-quality healthy rainbow trout pseudo-male fish, dissecting to obtain a pseudo-male fish spermary, placing the pseudo-male fish spermary in a clean anhydrous mortar, grinding the mortar to obtain a semen tissue mixed solution, filtering the mixed solution by using gauze to obtain pseudo-male fish semen, mixing the pseudo-male fish semen and a semen preservation solution according to the volume ratio of 1: 1, placing the mixture in an oxygen bag, controlling the oxygenation concentration to be 0.05-0.1 mmol/L, storing for 20-30 min, and taking out the mixture to be used for fish egg fertilization.
Compared with the prior proportion, the semen preservation solution increases the use of sodium citrate, the sodium citrate has excellent retarding performance and stability, is weak acid strong alkali salt, is a strong pH buffer agent, and plays a buffering role in reducing the concentration of electrolyte in the semen.
The method of the invention increases the use of oxygen, the oxygen can influence the survival rate of sperms, the integrity of membranes and the peroxidation of membrane lipids, a proper amount of oxygen is beneficial to sperm capacitation and acrosome reaction, however, high level of oxygen can cause oxidative stress, and the invention selects proper oxygen concentration to improve sperm capacitation to the maximum extent.
The semen preservation solution is prepared and placed at the temperature of 8-12 ℃ so as to reduce the temperature difference between semen and the semen preservation solution and avoid the stimulation of sperm due to temperature change, and the temperature of 8-12 ℃ is also the optimum temperature for oviposition and fertilization of the rainbow trout.
The invention has the beneficial effects that:
compared with the prior art, the invention increases the use of organic compounds, takes the buffering of the electrolyte concentration of the semen into consideration, improves the pH value of the semen, provides a good environment for the survival of the sperm, increases the utilization of oxygen in the process of accelerating the maturity of the sperm, and provides guarantee for improving the capacitation of the sperm to the maximum extent, which is not related in the prior art. The activation rate of the sperms of the pseudo-male fish obtained by the invention reaches more than 90%, and the fertilization rate and the eyeing rate of the fish eggs are both more than 70%, which are obviously higher than those of the eyed eggs obtained by the prior art, thereby greatly reducing the consumption of the fish eggs and reducing the waste of the fish eggs.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
Specific embodiment the method for improving the sperm utilization rate of the rainbow trout pseudo-male fish comprises the following steps:
, preparing a seminal fluid preserving fluid for the rainbow trout pseudo-male fish, wherein the seminal fluid preserving fluid comprises 6-8 g/L NaCl, 2-4 g/L KCl and 0.2-0.4 g/L CaCl2·2H2O、0.2～0.4g/L MgCl2·6H2O、0.1～0.3g/L NaHCO3、4～5g/LC6H5Na3O7And distilled water.
Secondly, after the semen preserving fluid is prepared, the pH value is adjusted to 9-10, and then the semen preserving fluid is placed for 2-3 hours at the temperature of 8-12 ℃ for standby.
Selecting high-quality healthy rainbow trout pseudo-male fish, dissecting to obtain a pseudo-male fish spermary, placing the pseudo-male fish spermary in a clean anhydrous mortar, grinding the pseudo-male fish spermary to obtain a semen tissue mixed solution, filtering the mixed solution by using gauze to obtain pseudo-male fish semen, and mixing the pseudo-male fish semen with a semen preservation solution according to a volume ratio of 1: 1, placing the mixture in an oxygen bag, controlling the oxygenation concentration to be 0.05-0.1 mmol/L, storing for 20-30 min, and taking out the mixture to be used for fish egg fertilization.
The second embodiment is different from in that the semen preservation solution in step comprises 7g/L NaCl, 3g/L KCl and 0.3g/L CaCl2·2H2O、0.3g/L MgCl2·6H2O、0.2g/L NaHCO3、4.5g/L C6H5Na3O7And distilled water the others are the same as in embodiment .
Third embodiment the difference between this embodiment and is that the semen preservation solution in step comprises 7.6g/L NaCl, 2.98g/L KCl, and 0.37g/L CaCl2·2H2O、0.31g/L MgCl2·6H2O、0.21g/LNaHCO3、4.2g/L C6H5Na3O7And distilled water the others are the same as in embodiment .
Fourth embodiment differs from embodiments -three in that in step two, the mixture is allowed to stand at 9-11 ℃ for 2-3 hours, and is otherwise the same as embodiments -three.
Fifth embodiment differs from embodiments -three in that in step two, the mixture is left at 10 ℃ for 2-3 hours, and is otherwise the same as embodiments -three.
Sixthly, this embodiment is different from of embodiments to fifths in that the oxygen concentration in step three is controlled to 0.06 to 0.09mmol/L, and is the same as of embodiments to fifths.
Seventhly, this embodiment is different from of embodiments to fifths in that the oxygen concentration in step three is controlled to 0.07 to 0.08mmol/L, and is the same as of embodiments to fifths.
Eighth embodiment differs from embodiments -seventeen in that the fish eggs can be fertilized after being stored for 25min in the third step, and is the same as embodiments -seventeen.
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1:
the method for improving the sperm utilization rate of the rainbow trout pseudo-male fish comprises the following steps:
, preparing a seminal fluid preserving fluid for the rainbow trout pseudo-male fish, wherein the seminal fluid preserving fluid is prepared from 7.6g/L NaCl, 2.98g/L KCl and 0.37g/L CaCl2·2H2O、0.31g/L MgCl2·6H2O、0.21g/L NaHCO3、4.2g/L C6H5Na3O7And distilled water.
Secondly, after the semen preserving fluid is prepared, the pH value of the solution is adjusted to 10 by NaOH, and then the solution is used after being placed for 3 hours at the temperature of 9 ℃, and the solution is prepared as it is.
Thirdly, selecting high-quality healthy rainbow trout pseudo-male fish, dissecting to obtain pseudo-male fish spermary, placing the pseudo-male fish spermary in a clean and anhydrous mortar, grinding the pseudo-male fish spermary by using the mortar, filtering out spermary tissue from the obtained semen spermary tissue mixture through gauze, and collecting the pseudo-male fish semen and the semen preservation solution according to the volume ratio of 1: 1, mixing, placing in an oxygen bag, oxygenating to a concentration of 0.05mmol/L, and fully contacting for 30 min. The obtained semen is activated by BSS (physiological saline), and the activation rate of the sperms is observed under a microscope and reaches more than 70 percent.
Obtaining 2L of rainbow trout roes by an artificial egg collection method, washing off impurities and egg skins by BSS (normal saline), taking 10 ml of semen mixed solution, fully and uniformly stirring the semen mixed solution and the roes, activating by BSS (normal saline), standing for 3 minutes, washing fertilized eggs by clear water, and placing the fertilized eggs in an incubation barrel for incubation by flowing water.
The method for improving the sperm utilization rate is used as a control, and comprises the following specific steps:
, preparing a seminal fluid preserving fluid for the rainbow trout pseudo-male fish, wherein the seminal fluid preserving fluid is prepared from 7.6g/L NaCl, 2.98g/L KCl and 0.37g/L CaCl2·2H2O、0.31g/L MgCl2·6H2O、0.21g/L NaHCO3And distilled water.
Secondly, after the semen preserving fluid is prepared, the pH value of the solution is adjusted to 10 by using NaOH, and the solution is prepared as it is used.
Thirdly, selecting high-quality healthy rainbow trout pseudo-male fish, dissecting to obtain pseudo-male fish spermary, placing the pseudo-male fish spermary in a clean and anhydrous mortar, grinding the pseudo-male fish spermary by using the mortar, filtering out spermary tissue from the obtained semen spermary tissue mixture through gauze, and collecting the pseudo-male fish semen and the semen preservation solution according to the volume ratio of 1: 1 mixing, activating the obtained semen with BSS (physiological saline), and observing the activation rate of the sperms under a microscope to reach more than 30 percent.
Obtaining 2L of rainbow trout roes by an artificial egg collection method, washing off impurities and egg skins by BSS (normal saline), taking 20 ml of semen mixed solution, fully and uniformly stirring the semen mixed solution and the roes, activating by BSS (normal saline), standing for 3 minutes, washing fertilized eggs by clear water, and placing the fertilized eggs in an incubation barrel for incubation by flowing water.
The effect of different treatments on sperm and zygote hatching is shown in table 1.
TABLE 1 Effect of different treatments on sperm and fertilized egg hatching
Group of Total egg number L Activation rate% Fertilization rate% Percentage of eye formation% The floating rate%
Prior Art 5 60％ 50％ 56％ 50％
Improved method 5 90％ 72％ 72％ 65％
Normal seminal fluid 5 95％ 75％ 80％ 68％
The data show that the activation rate, the fertilization rate and the eye forming rate of the fish roe processed by the method are obviously higher than those of the prior art, the consumption of the fish roe is greatly reduced, and the waste of the fish roe is reduced.

Claims (4)

1, method for improving the sperm utilization rate of rainbow trout pseudo-male fish, which is characterized in that the method comprises the following steps:
, preparing semen of rainbow troutThe semen preserving solution comprises 7g/L NaCl, 3g/L KCl and 0.3g/LCaCl2·2H2O、0.3g/L MgCl2·6H2O、0.2g/L NaHCO3、4.5g/L C6H5Na3O7And distilled water;
secondly, after the semen preserving fluid is prepared, adjusting the pH value to 9-10, and then placing for 2-3 hours at 10 ℃ for later use;
selecting high-quality healthy rainbow trout pseudo-male fish, dissecting to obtain a pseudo-male fish spermary, placing the pseudo-male fish spermary in a clean anhydrous mortar, grinding the mortar to obtain a semen tissue mixed solution, filtering the mixed solution by using gauze to obtain pseudo-male fish semen, mixing the pseudo-male fish semen and a semen preservation solution according to the volume ratio of 1: 1, placing the mixture in an oxygen bag, controlling the oxygenation concentration to be 0.05-0.1 mmol/L, storing for 20-30 min, and taking out the mixture to be used for fish egg fertilization.
2. The method for improving the sperm utilization rate of rainbow trout pseudo-male fish of claim 1, wherein the oxygenation concentration in the third step is controlled to be 0.06-0.09 mmol/L.
3. The method for improving the sperm utilization rate of rainbow trout pseudo-male fish of claim 1, wherein the oxygenation concentration in the third step is controlled to be 0.07-0.08 mmol/L.
4. The method for improving the sperm availability of rainbow trout pseudo-males as claimed in claim 1, wherein the rainbow trout is stored for 25min in step three and then taken out for fertilization of roes.
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CN102792910B (en) * 2012-08-31 2014-03-05 北京市水产科学研究所 Cultivating method of oncorhynchus mykiss walbaum gynoecial hybrid offspring seed
CN102972326B (en) * 2012-12-28 2014-01-01 中国水产科学研究院黑龙江水产研究所 Method for breeding Oncorhynchus masou masou
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CN102960279A (en) * 2012-11-30 2013-03-13 射阳康余水产技术有限公司 Artificial insemination method for yellow-head catfish in hypotonic solution
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