CN114208813A - Cow frozen semen diluent, preparation method thereof and semen freezing method - Google Patents
Cow frozen semen diluent, preparation method thereof and semen freezing method Download PDFInfo
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- CN114208813A CN114208813A CN202111591719.3A CN202111591719A CN114208813A CN 114208813 A CN114208813 A CN 114208813A CN 202111591719 A CN202111591719 A CN 202111591719A CN 114208813 A CN114208813 A CN 114208813A
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The invention discloses a frozen cow semen diluent, a preparation method thereof and a semen freezing method, wherein the frozen cow semen diluent is prepared from egg yolk, sodium citrate, fructose, Tris, glycerol, streptomycin, cysteamine, reductive glutathione, taurine, disodium adenosine triphosphate, seminal serum and water-soluble laminarin and is used for semen freezing, the obtained frozen semen is unfrozen and conforms to the national standard GB4143-2008, wherein the egg yolk, the sodium citrate, the fructose, the Tris, the glycerol and the streptomycin are used as basic liquid, and the cysteamine, the reductive glutathione, the taurine, the disodium adenosine triphosphate, the seminal serum and the water-soluble laminarin are used as two liquid, so that the damage of freezing to sperm mitochondria can be protected, and the survival time of the sperm can be prolonged. The frozen semen diluent for the dairy cattle can ensure that the sperm can preserve mitochondrial energy to the maximum extent after freezing and thawing and prolong the survival time. Through artificial insemination conception rate tests, the conception rate of the young cattle in the estrus is improved by 1.9 percent, and the conception rate of the young cattle in the estrus is improved by 3.1 percent.
Description
Technical Field
The invention belongs to the field of livestock breeding, and particularly relates to a frozen semen diluent for dairy cows, a preparation method thereof and a semen freezing method.
Background
In foreign countries, the developed countries of the dairy industry have made great progress in the aspects of frozen diluent, frozen semen production process, physiology and genetic selection of frozen semen yield and the like. The activity of frozen semen after freezing is generally more than 0.4. In recent years, through continuous efforts of domestic bull stations, the production process flow of the normalized frozen bull semen is initially established in China.
But the conception rate of the frozen cow semen after artificial fertilization has not been greatly broken through all the time, young cows are maintained at about 50-55%, and multiparous cows are maintained at about 35-40%, and the pregnancy rate is reduced along with the increase of milk yield of cows. The main reason for this is that the freezing process takes about 6 hours, and the lipid peroxidation during the freezing process reduces the sperm quality and results in the loss of the sperm motility of 40-50%. Due to the interaction of various factors from the outside, the number of dead and damaged sperm is increased, resulting in the generation of excessive reactive oxygen species ROS. Both cold stress and freeze damage to sperm are caused by the generation of excessive reactive oxygen and oxygen stress, and peroxidative damage is a significant cause of sperm degradation after freeze-thaw. Under normal physiological conditions, the ROS of the sperms is in a dynamic equilibrium state, and the balance state is broken because the sperms are contacted with the external environment in the process of freezing and unfreezing the semen, so that the ROS of the sperms is increased, the fluidity of a sperm membrane is changed, mitochondria and DNA are damaged, and the quality of the semen is reduced. A small amount of acrosome swelling, separation, rupture and other malformed sperms also exist in the fresh semen, and after freezing and thawing, part of the sperms can suffer from acrosome swelling, separation and rupture, the head and the tail of the sperms have cleavage, deoxyribonucleic acid is reduced, the lipoprotein membrane has high permeability, and protein is damaged, so the sperms do not have insemination capability any more. The sperm acrosome membrane is considered to be the most vulnerable key structure in a low temperature environment. The freezing process affects the normal flow and structural integrity of the plasma membrane, greatly reducing the fertilization ability of the sperm. The freeze-thaw process can cause phenomena such as enhanced permeability of sperm cell membranes, loss of cholesterol on the surface of sperm plasma membranes, and reduced sperm motility.
Therefore, in order to improve the conception rate of the frozen bovine semen after artificial insemination, further improvement in the quality of the frozen semen is required.
Disclosure of Invention
In order to solve the technical problems, the invention provides a frozen semen diluent for dairy cows, a preparation method thereof and a semen freezing method, wherein semen is frozen by adopting the frozen semen diluent for the dairy cows, and the conception rate of the obtained frozen semen is improved by 1.9% in the estrus of the young cattle and 3.1% in the estrus of the born cattle after the unfrozen frozen semen is tested by artificial insemination, so that the problem of low conception rate of the traditional frozen semen of the dairy cows after artificial insemination is solved.
The frozen semen diluent for the dairy cow comprises the following components in every 100 mL:
20mL of yolk, 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 15 ten thousand IU of streptomycin, 75-80mg of cysteamine, 0.23-0.27g of reductive glutathione, 9.5-10.5mg of taurine, 3.5-5mg of disodium adenosine triphosphate, 2-2.5mL of refined white, 0.45-0.60mg of water-soluble laminarin and the balance of distilled water;
the pH value of the cow frozen semen diluent is 6.8-7.
Preferably, the essence is bovine essence.
The invention also provides a preparation method of the cow frozen semen diluent, which comprises the following steps: adding 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 75-80mg of cysteamine, 0.23-0.27g of reducing glutathione, 9.5-10.5mg of taurine, 3.5-5mg of disodium adenosine triphosphate and 0.45-0.60mg of water-soluble laminarin into distilled water, stirring uniformly, placing in a 75-80 ℃ water bath kettle for disinfection for 30min, cooling to normal temperature, then adding egg yolk, streptomycin and refined liquid, and using distilled water to fix the volume to 100mL, and adjusting the pH value to 6.8-7.
The invention also provides a method for diluting frozen cow semen to freeze cow semen by using the dilution liquid, which comprises the following steps:
a) semen dilution
Adding the semen diluent into the semen to dilute the cow semen until the number of the semen in the solution reaches 2.4 multiplied by 106Per mL;
b) temperature reduction
Transferring the diluted semen into a thin tube, sealing, placing in a refrigerator at 4 deg.C, cooling, and balancing for 3-5 h;
c) freezing
Placing the balanced tubule semen stacking drawer in a full-automatic freezer, freezing, and finally placing in liquid nitrogen for preservation;
d) packaging frozen semen;
and (5) packaging and warehousing the frozen semen of the thin tube.
Preferably, in step c), the freezing comprises three stages, in particular:
in the first stage, the temperature of a partition plate before freezing is 23 ℃, and the temperature is reduced to 0-4 ℃ within 5 hours;
in the second stage, the freezing temperature is reduced to 0 to minus 5 ℃ at the cooling speed of 0.15 ℃/s, and the temperature is kept for 120 s;
and in the third stage, the freezing temperature is reduced to-5 to-130 ℃ at the cooling rate of 0.66 ℃/s, and is kept for 180 s.
The invention provides a frozen cow semen diluent, a preparation method thereof and a semen freezing method, wherein the frozen cow semen diluent is prepared from egg yolk, sodium citrate, fructose, Tris, glycerol, streptomycin, cysteamine, reductive glutathione, taurine, disodium adenosine triphosphate, seminal serum and water-soluble laminarin and is used for semen freezing, the obtained frozen semen is unfrozen and conforms to the national standard GB4143-2008, wherein the egg yolk, the sodium citrate, the fructose, the Tris, the glycerol and the streptomycin are used as basic liquid, and the cysteamine, the reductive glutathione, the taurine, the disodium adenosine triphosphate, the seminal serum and the water-soluble laminarin are used as two liquid, so that the damage of freezing to sperm mitochondria can be protected, and the survival time of the sperm can be prolonged. The frozen semen diluent for the dairy cattle can ensure that the sperm can preserve mitochondrial energy to the maximum extent after freezing and thawing and prolong the survival time. Through artificial insemination conception rate tests, the conception rate of the young cattle in the estrus is improved by 1.9%, and the conception rate of the young cattle in the estrus is improved by 3.1%, so that the artificial insemination conception rate test method has popularization and application values.
Drawings
FIG. 1 is a diagram of a computer aided analyzer inspection interface;
FIG. 2 is a mitochondrial TEM image of fresh sperm;
FIG. 3 is a TEM image of mitochondria of frozen sperm of Experimental group 1;
FIG. 4 is a graph of sperm capacitation with fluorescent dye detection;
FIG. 5 is a graph of ATP detection standards.
Detailed Description
The invention provides a cow frozen semen diluent, wherein each 100mL of the diluent comprises the following components:
20mL of yolk, 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 15 ten thousand IU of streptomycin, 75-80mg of cysteamine, 0.23-0.27g of reductive glutathione, 9.5-10.5mg of taurine, 3.5-5mg of disodium adenosine triphosphate, 2-2.5mL of refined white, 0.45-0.60mg of water-soluble laminarin and the balance of distilled water;
the pH value of the cow frozen semen diluent is 6.8-7.
According to the invention, yolk, sodium citrate, fructose, Tris, glycerol and streptomycin are used as base liquid, and cysteamine, reductive glutathione, taurine, disodium adenosine triphosphate, refined liquid and water-soluble laminarin are used as two liquid liquids, so that damage of freezing to sperm mitochondria can be protected, and the survival time of the sperm is prolonged. The frozen semen diluent for the dairy cattle can ensure that the sperm can preserve mitochondrial energy to the maximum extent after freezing and thawing and prolong the survival time. Through artificial insemination conception rate tests, the conception rate of the young cattle in the estrus is improved by 1.9%, and the conception rate of the young cattle in the estrus is improved by 3.1%, so that the artificial insemination conception rate test method has popularization and application values.
The yolk can protect sperm from freezing and promote the combination of sperm and transparent zone of egg cell. In the invention, the addition amount of the yolk in each 100mL of the cow frozen semen diluent is 20 mL. In the present invention, the source of the yolk is not limited, and the yolk may be generally commercially available.
The sodium citrate can prevent the change of pH in the semen processing process, and has good buffering effect on metabolic acidosis and enzyme activity reaction. In the invention, the addition amount of the sodium citrate in the cow frozen semen diluent of every 100mL is 2.32 g. In the present invention, the source of sodium citrate is not limited, and it is generally available on the market.
The fructose provides nutrients for the in vitro metabolism of the sperms and supplements the energy consumed by the sperms. In the invention, the addition amount of fructose in the cow frozen semen diluent of every 100mL is 0.5 g. The fructose source in the present invention is not limited, and may be commercially available.
Tris is used as a buffer substance, and can buffer the pH value of semen, adjust osmotic pressure and create a proper environment for sperms. In the present invention, the amount of Tris added per 100mL of the cow frozen semen diluent is 2.42 g. In the present invention, the source of Tris is not limited, and may be generally commercially available.
The glycerol is used as the best anti-freezing protective agent in the ultralow temperature freezing process of sperms, has the characteristic of water solubility, is not easy to crystallize, and has the characteristics of strong water absorption and capability of reducing the freezing point of an aqueous solution. In the present invention, the amount of glycerin added per 100mL of the cow frozen semen diluent is 5.4 g. In the present invention, the source of glycerin is not limited, and glycerin is generally available on the market.
Penicillin and streptavidin are used to control pathogenic and non-pathogenic diseases in semen in the production of capillary frozen semen, and the penicillin and the streptavidin have the characteristics of wide antibacterial range, low cost and the like. According to the antibiotic dosage in the semen diluent formula, the estrus conception rate of the cow is improved by mainly controlling venereal diseases and autism. In the present invention, the amount of streptomycin in the frozen semen diluent per 100mL of cows is 15 ten thousand IU. In the present invention, the source of streptomycin is not limited, and the streptomycin is generally commercially available.
The cysteamine and the reduced glutathione are both used as nutritional agents, and can provide nutrition for sperms and prolong the survival time of the sperms. In the invention, the addition amount of the cysteamine in the cow frozen semen diluent of every 100mL is 75-80mg, and the addition amount of the reducing glutathione is 0.23-0.27 g; preferably, the addition amount of cysteamine is 78mg and the addition amount of reduced glutathione is 0.25 g. In the present invention, the sources of cysteamine and reduced glutathione are not limited, and they may be commercially available.
The taurine is used for inhibiting freezing damage of the frozen diluent of the dairy cow and improving the activity of mitochondria. In the invention, the addition amount of taurine in every 100mL of the cow frozen semen diluent is 9.5-10.5 mg; preferably, taurine is added in an amount of 10 mg.
The disodium adenosine triphosphate can protect the sperm mitochondria and avoid damage to the sperm mitochondria caused by freezing. In the invention, the adding amount of the adenosine disodium triphosphate in each 100mL of frozen semen diluent of the dairy cattle is 3.5-5 mg; preferably, the amount of disodium adenosine triphosphate added is 4 mg. In the present invention, the source of the disodium adenosine triphosphate is not limited, and the disodium adenosine triphosphate may be generally commercially available.
The semen can prolong the survival time of semen and improve conception rate. In the invention, the addition amount of the semen in each 100mL of the cow frozen semen diluent is 2-2.5 mL; preferably, the amount of added fine powder is 2.2 mL. The invention is characterized in that the semen is bovine semen, which is extracted from the semen of bulls, and the extraction method is obtained according to the conventional method in the field, namely, the semen of bulls is centrifuged, and the obtained supernatant is the semen.
The water-soluble laminarin can prolong the survival time of sperms and improve the conception rate. In the present invention, the amount of the spermine added per 100mL of the cow frozen semen dilution is 0.45-0.60mg, preferably, the spermine is 0.50 mg. The source of the water-soluble laminarin is not limited in the invention, and the laminarin can be generally sold in the market.
The invention also provides a preparation method of the cow frozen semen diluent, which comprises the following steps: adding 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 75-80mg of cysteamine, 0.23-0.27g of reducing glutathione, 9.5-10.5mg of taurine, 3.5-5mg of disodium adenosine triphosphate and 0.45-0.60mg of water-soluble laminarin into distilled water, stirring uniformly, placing in a 75-80 ℃ water bath kettle for disinfection for 30min, cooling to normal temperature, then adding egg yolk, streptomycin and refined liquid, and using distilled water to fix the volume to 100mL, and adjusting the pH value to 6.8-7.
Wherein, yolk, sodium citrate, fructose, Tris, glycerol, streptomycin, cysteamine, reductive glutathione, taurine, adenosine disodium triphosphate, refined white and water-soluble laminarin are all the same as the above, and are not repeated herein.
Compared with the conventional frozen semen diluent, the frozen semen diluent for the dairy cattle prepared by the invention can prolong the survival time of the semen in vitro after the semen is frozen and thawed for 6-8 hours; through artificial insemination tests, the conception rate of young cattle in the estrus is improved by 1.9%, and the conception rate of the young cattle in the estrus is improved by 3.1%, so that the artificial insemination method has popularization and application values.
Attention is required in preparing frozen semen: all tools used in the preparation process need to be thoroughly washed and disinfected for use; 2. the pH value of the used distilled water is neutral; 3. all the components need to be weighed accurately and dissolved sufficiently, and are disinfected after being filtered and sealed.
The invention also provides a semen freezing method for the cow frozen semen diluent, which comprises the following steps:
a) semen dilution
Adding the semen diluent into the semen to dilute the cow semen until the number of the semen in the solution reaches 2.4 multiplied by 106Per mL;
b) temperature reduction
Transferring the diluted semen into a thin tube, sealing, placing in a refrigerator at 4 deg.C, cooling, and balancing for 3-5 h;
c) freezing
Placing the balanced tubule semen stacking drawer in a full-automatic freezer, freezing, and finally placing in liquid nitrogen for preservation;
d) packaging frozen semen;
and (5) packaging and warehousing the frozen semen of the thin tube.
When the diluent of the frozen semen of the dairy cattle is used for semen freezing, the conception rate of the obtained frozen semen can be improved after the frozen semen is unfrozen, and through an artificial insemination test, the conception rate of young cattle in the estrus is improved by 1.9 percent, and the conception rate of the young cattle in the estrus is improved by 3.1 percent.
In an embodiment of the invention, in step c), the freezing comprises three stages, in particular:
in the first stage, the temperature of a partition plate before freezing is 23 ℃, and the temperature is reduced to 0-4 ℃ within 5 hours;
in the second stage, the freezing temperature is reduced to 0 to minus 5 ℃ at the cooling speed of 0.15 ℃/s, and the temperature is kept for 120 s;
and in the third stage, the freezing temperature is reduced to-5 to-130 ℃ at the cooling rate of 0.66 ℃/s, and is kept for 180 s.
When the cow frozen semen diluent is used for freezing semen, attention needs to be paid to the following steps: 1. the frozen semen diluent of the dairy cow needs to be kept fresh, and is preferably used as it is, if the conditions do not allow, the frozen semen diluent can be stored in a refrigerator for 1 week; 2. after semen collection, semen should be diluted immediately, so as to avoid the semen from being stored for a longer time, and the number of the semen is increased; 3. the temperature of the diluent and the temperature of the semen must be adjusted to be consistent, so as to avoid the damage to the sperm caused by overlarge temperature difference; 4. during dilution, the diluent is slowly added along the glass rod, so that the diluent is prevented from being rapidly poured into semen to cause local dilution and striking; 5. after dilution, the semen collection cup is slightly rotated to uniformly mix semen and the diluent without violent shaking.
To further illustrate the present invention, the following examples are provided for illustration.
Example 1
The frozen semen diluent for the dairy cow comprises the following components in every 100 mL:
20mL of yolk, 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 15 ten thousand IU of streptomycin, 78mg of cysteamine, 0.25g of reductive glutathione, 10mg of taurine, 4mg of adenosine disodium triphosphate, 2.3mL of bovine serum, 0.50mg of water-soluble laminarin and the balance of distilled water.
Adding the sodium citrate, fructose, Tris, glycerol, cysteamine, reductive glutathione, taurine, disodium adenosine triphosphate and water-soluble laminarin into distilled water, stirring uniformly, placing in a water bath kettle at 80 ℃ for disinfection for 30min, cooling to normal temperature, adding the yolk, the streptomycin and the taurine into the water bath kettle, fixing the volume to 100mL by using the distilled water, and adjusting the pH value to 7.
Example 2
The frozen semen diluent for the dairy cow comprises the following components in every 100 mL:
20mL of yolk, 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 15 ten thousand IU of streptomycin, 75-80mg of cysteamine, 0.23g of reductive glutathione, 9.5mg of taurine, 3.5mg of adenosine disodium triphosphate, 2mL of bovine serum, 0.45mg of water-soluble laminarin and the balance of distilled water.
Adding the sodium citrate, fructose, Tris, glycerol, cysteamine, reductive glutathione, taurine, disodium adenosine triphosphate and water-soluble laminarin into distilled water, stirring uniformly, placing in a 75 ℃ water bath kettle for disinfection for 30min, cooling to normal temperature, then adding the yolk, the streptomycin and the taurine, diluting to 100mL with distilled water, and adjusting the pH value to 6.8.
Example 3
The frozen semen diluent for the dairy cow comprises the following components in every 100 mL:
20mL of yolk, 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 15 ten thousand IU of streptomycin, 75-80mg of cysteamine, 0.27g of reductive glutathione, 10.5mg of taurine, 5mg of adenosine disodium triphosphate, 2.5mL of bovine serum, 0.60mg of water-soluble laminarin and the balance of distilled water.
Adding the sodium citrate, fructose, Tris, glycerol, cysteamine, reductive glutathione, taurine, disodium adenosine triphosphate and water-soluble laminarin into distilled water, stirring uniformly, placing in a water bath kettle at 80 ℃ for disinfection for 30min, cooling to normal temperature, adding the yolk, the streptomycin and the taurine into the water bath kettle, fixing the volume to 100mL by using the distilled water, and adjusting the pH value to 67.
Comparative example 1
The frozen semen diluent for the dairy cow comprises the following components in every 100 mL:
20mL of yolk, 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 15 ten thousand IU of streptomycin and the balance of distilled water.
Adding the sodium citrate, the fructose, the Tris and the glycerol into distilled water, stirring uniformly, placing in a water bath kettle at 80 ℃ for disinfection for 30min, cooling to normal temperature, adding the yolk and the streptomycin, adding distilled water to a constant volume of 100mL, and adjusting the pH value to 7.
Comparative example 2
Based on the example 1, the cow frozen semen diluent does not contain cysteamine, and the rest components, the proportion and the method are the same as the example 1.
Comparative example 3
Based on the example 1, the diluent of the frozen semen of the dairy cow has no reductive glutathione, and the rest components, the proportion and the method are the same as the example 1.
Comparative example 4
Based on the example 1, the dilution of the frozen semen of the dairy cow does not contain taurine, and the rest components, the proportion and the method are the same as the example 1.
Comparative example 5
Based on the example 1, the dilution of the frozen semen of the cow has no adenosine disodium triphosphate, and the other components, the proportion and the method are the same as the example 1.
Comparative example 6
Based on the example 1, the diluent of the frozen semen of the dairy cow has no semen and the other components, the proportion and the method are the same as the example 1.
Comparative example 7
Based on the example 1, the diluent of the frozen semen of the dairy cow has no water-soluble laminarin, and the other components, the proportion and the method are the same as the example 1.
Preparation of frozen semen
Respectively designing experimental groups 1-3 and comparison groups 1-7, wherein the experimental groups 1-3 are obtained by freezing semen by adopting the cow frozen semen diluent obtained in the embodiment 1-3 to obtain frozen semen; and the comparison groups 1 to 7 adopt the frozen semen diluent obtained in the comparison groups 1 to 7 to carry out semen freezing to obtain frozen semen.
The semen freezing method of the frozen semen diluent of the dairy cow comprises the following steps:
a) semen dilution
Bull semen is collected by a false vagina method, stood on the right back side of the livestock, held by the false vagina with the right hand and with one end of the semen collecting cup upward. When the bull climbs, the false vagina and the penis of the bull are in a straight line, the penis is guided into the entrance of the false vagina, the bull drives the false vagina backwards to rush forwards, namely ejaculation is carried out, and then the false vagina semen collection cup is inclined downwards, so that semen completely flows into the semen collection cup. When the bull climbs down, the false vagina is held to move backwards along with the penis, the switch is turned on, air is discharged, the false vagina is rapidly and naturally taken down after the penis is automatically softened and pulled out, and the false vagina is immediately sent into the semen processing chamber to detect the sperm motility and the deformity rate, the fresh semen sperm motility is more than or equal to 65 percent, and the deformity rate is less than or equal to 15 percent, and further, the bull semen conforms to GB 4143-2008.
The cow frozen semen diluents of examples 1 to 3 and comparative examples 1 to 7 were added to bull semen to dilute the bull semen until the number of sperm in the solution reached 2.4X 106Per mL;
b) temperature reduction
Transferring the diluted bull semen into a thin tube, sealing, placing in a refrigerator at 4 ℃, and cooling and balancing for 4 hours;
c) freezing
Placing the balanced tubule semen stacking drawer in a full-automatic freezer, freezing, and finally placing in liquid nitrogen for preservation; wherein freezing includes three stages, specifically:
the first stage, the temperature of the clapboard before freezing is 23 ℃, and the temperature is slowly reduced to 0 ℃ within 5 h;
in the second stage, the freezing temperature is reduced to-5 ℃ at the cooling speed of 0.15 ℃/s, and the temperature is kept for 120 s;
in the third stage, the freezing temperature is reduced to-130 ℃ at the cooling rate of 0.66 ℃/s, and the temperature is kept for 180 s;
d) packaging frozen semen;
and (5) packaging and warehousing the frozen semen of the thin tube.
First, cryoinjury study
1.1, Manual detection
Manual detection: the sperm in the frozen sperm obtained from the experimental groups 1-3 and the comparative groups 1-7 were detected, and the results are shown in Table 1.
The frozen semen obtained from the experimental groups 1-3 and the comparative groups 1-7 is unfrozen according to GB4143-2008, the frozen semen is unfrozen in a water bath kettle at 37 ℃, and the integrity rate of a plasma membrane, the integrity rate of a acrosome, the integrity rate of DNA, the content of ATP and the activity of mitochondria are detected after unfreezing.
Detection of plasma membrane integrity:
the thawed semen of experimental group 1-3 and comparative group 1-7 was washed with PBS for 2 times and centrifuged (2000rpm, 5min) to adjust the sperm density in PBS suspension to 7.5 × 106one/mL.
1.0mmol/L PI 5. mu.L is added into the corresponding 400. mu.L LPBS suspensions of the experimental group 1-3 and the comparative group 1-7, and the mixture is incubated at 37 ℃ for 15min and then subjected to computer-aided analyzer detection (see figure 1), and the detection results are shown in Table 1. The sperm stained with PI in red fluorescence is representative of dead sperm and can be seen in FIG. b, FIG. 1b is a graph of the plasma membrane integrity rate of the experimental group 1, and the black arrows in FIG. 1b indicate red fluorescence.
And (3) detecting the acrosomal integrity:
5 μ L of 1.0mmol/L FITC-PNA was placed in a container containing 7.5X 106The sperm/mL is incubated in 400 mu LPBS solution at 37 ℃ for 30min and then detected by a computer-aided analyzer, and green fluorescence indicates sperm with damaged acrosomal membrane.
Sperm capacitation detection:
first, 750. mu. mol/LCTC was dissolved in a buffer containing 20. mu. mol/L Trise base and 130. mu. mol/L NaCl (pH 7.8), followed by filtration. Mixing 15 mu of LCTC with 10 mu of semen, adding 0.5 mu of 12.5% (v/v) glutaraldehyde solution for fixation, observing under fluorescence microscope blue fluorescence (excitation light: 400-: type F: the head of the whole sperm has uniform fluorescence which is the sperm with incompetence and complete acrosome; type B: in the posterior area of the acrosome of the sperm, the part close to the tail part has no fluorescence or very weak fluorescence, while the anterior part of the sperm has uniform fluorescence and is the sperm with capacitation and complete acrosome; ③ AR type: the head of the whole sperm has no fluorescence or very weak fluorescence, and is a sperm with an incomplete acrosome, that is, a sperm with acrosome reaction or a sperm with acrosome defect, as shown in fig. 4, fig. 4 is a sperm capacitation detection chart of the experimental group 1, wherein a is F type, B is B type, and C is AR type.
Detection of DNA integrity:
the above PBS suspension (7.5X 10) was subjected to the SCD method using a sperm DNA fragment detection kit (Shenzhen Boruide Biotech Co., Ltd.)6piece/mL) was mixed with agarose and spread on a glass slide for acid treatment, then cells were lysed to remove nuclear protein, stained with a staining agent, and the results were finally observed with a microscope. Evaluation was based on the halo produced by sperm DNA, with normal sperm DNA producing a diffuse halo and damaged sperm DNA producing no or minimal halo. Counting 500 sperms in multiple visual fields, and recording the proportion of different halo spermsAnd calculating the DNA integrity rate.
Detection of mitochondrial Activity:
the PBS suspension was taken (7.5X 10)6mL)200ul, centrifuging (2000rpm, 5min), collecting sperm, adding 500ul JC-1 working solution, mixing, standing at 37 deg.C and 5% CO2Incubate 20min in incubator, centrifuge at room temperature (2000rpm, 5min) to collect sperm, wash twice with 1 × Incubation Buffer, resuspend and precipitate sperm 500ul 1 × Incubation Buffer, mix well, detect by flow cytometry, measure 10000 sperm per sample. JC-1 monomer and polymer fluorescence signals were obtained on FL1 and FL2 detectors, respectively, JC-1 polymer (E)x＝488nm，Em575nm) shows high energy mitochondria with red fluorescence, JC-1 monomer (E)x＝488nm，Em575nm) showed green fluorescence indicating low energy mitochondria and was detected using a computer-assisted analyzer.
The observation of mitochondria and acrosomes of sperm by TEM and the results are shown in FIG. 2 and FIG. 3 (wherein FIG. 2 is TEM image of fresh semen, FIG. 3 is TEM image of experimental group 1, FIG. 2a is 20000 times magnified, FIG. 2b is 25000 times magnified, FIG. 2c and FIG. 2d are 40000 times magnified, FIG. 2d is 50000 times magnified, FIG. 3a is 30000 times magnified, FIG. 3b and FIG. 3c are 40000 times magnified, and FIG. 3d and FIG. 3e are 50000 times magnified), and the following steps are performed:
the PBS suspension was taken (7.5X 10)6mL) daughter cells were washed with PBS 2.5% (M/V) glutaraldehyde 0.1M arsenate buffer at 4 ℃ overnight and washed with DPBS, then 1% osmium acid (osmium tetraoxide) fixed DPBS washed, 30%, 50%, 70%, 80%, 90%, 100% acetone gradient dehydrated epoxy resin SPURR embedded-polymerized LKB-V microtome section ultrathin section thickness 50-nm (diamond knife) electron microscope copper mesh double stained with uranium acetate-lead citrate sample loaded copper mesh was placed in a glass dish and dried for overnight Transmission Electron Microscope (TEM) to acquire images at 80k (HITACHI, NewBio-TEMH-7500, Japan).
And (3) detection of ATP content:
the luciferase-based Biyun ATP content detection kit is used for detecting the ATP content of sperms, the operational steps refer to the Biyun mitochondrial membrane potential detection kit, and the following test conditions are optimized:
preparation of atp standard curve: respectively diluting ATP standard solution (0.5mmol/L) into low-concentration standard solution of 0.01. mu. mol/L, 0.05. mu. mol/L, 0.1. mu. mol/L, 0.5. mu. mol/L, 1. mu. mol/L and 5. mu. mol/L, and temporarily storing on ice;
preparing ATP detection working solution: diluting the ATP detection reagent by 10 times by using ATP detection lysate, and placing the diluted ATP detection reagent on ice for temporary storage;
C. adding 200 μ L ATP detection lysate into the 400 μ LPBS suspension, resuspending, mixing with vortex, and lysing on ice for 30 min;
D. after the cracking is finished, 12000g and centrifugation is carried out for 10min at 4 ℃, and supernatant is collected and temporarily stored on ice;
E. assay samples were added to 6-well plates according to the following table:
F. detecting the autofluorescence intensity by using a multifunctional microplate reader, and finishing and analyzing the test result by using Excel software to obtain an ATP detection standard curve shown in figure 5;
the rest steps are the same as the steps for detecting the ATP content by the kit, and are not described in detail herein.
TABLE 1 results of artificial sperm freezing damage
As can be seen from table 1 and fig. 2-3;
1) the freezing can damage the plasma membrane of the sperm, and the frozen plasma membrane protein of the sperm is aggregated to influence the combination of a membrane receptor and a ligand. The proportion of frozen sperm with intact DNA in the experimental and comparative groups 1-3 and 1-7 was significantly lower than fresh sperm, indicating that genetic material carried in the sperm nuclei was damaged after freezing and thawing, which may cause early embryonic death.
2) The proportion of acrosome-reacted sperm after freezing was significantly higher in experimental and comparative groups 1-3 and 1-7 compared to fresh sperm (table 1), and as can be seen from electron microscope sections taken through the acrosome, the freezing caused acrosome reaction, the sperm showed a capacitation change, and the acrosome content disappeared (fig. 2-3). This variation is different from true capacitation and acrosome reactions prior to fertilization, and sperm freezing and thawing occurring in this variation may present fertilization dysfunction
3) The proportion of sperm having high-energy mitochondria after freezing was significantly reduced in the experimental group 1-3 and the comparative group 1-7 compared to fresh sperm (table 1). After being frozen, the sperm mitochondria are irregularly arranged and have different densities, the mitochondria are overlapped in multiple layers and surround the tail inner shaft silk, and part of the mitochondria are cystic expanded. The crossing mechanism above it is significantly reduced, mitochondrial membranes are thickened etc. (fig. 2-3). The damage is caused by the fact that when sperm cells are subjected to freeze thawing, ice crystals are formed in and among the cells and are melted to cause cell damage, and the influence on biological membranes (such as cell membranes, Golgi membranes and mitochondrial membranes) is more obvious due to the toxic action of cryoprotectants. Membrane proteins on the cell membrane denature, altering the interaction between membrane lipids and proteins. The permeability of the membrane is changed, the permeability of sodium ions and potassium ions is increased, potassium ions and calcium ions are replaced, the stability of the membrane is reduced, and the charges on the surface of the cell are changed. The enzyme activity in the plasma membrane and the cells decreases. Mitochondrial membrane disruption, oxidative phosphorylation coupling, energy metabolism disorder, and reduced energy supply.
4) The plasma membrane integrity, acrosome integrity, DNA integrity, ATP content and mitochondrial activity of the frozen semen of the experimental groups 1-3 are all higher than those of the comparative groups 1-7. Therefore, the frozen semen diluent for the dairy cattle can ensure that the sperms can maximally preserve the plasma membrane integrity rate, acrosome integrity rate, DNA integrity rate, ATP content and mitochondrial activity after being frozen and thawed.
Second, in vitro validation
2.1, in vitro verification method: and (3) simulating the reproductive tract environment of the female animals, unfreezing 10 frozen semen in each group according to the GB4143-2008 requirement, uniformly mixing the unfrozen semen, and averagely distributing the mixture into 4 centrifugal tubes of 1.5 mL. Placing each set of centrifuge tubes with 5% CO by volume2The frozen semen was cultured in a carbon dioxide incubator at 38.5 ℃ under saturated humidity and observed for viability under a microscope, and the viability of the frozen semen and the prolonged sperm survival time were measured at 0h, 12h, 18h and 24h, respectively (see Table 2 for results).
TABLE 2 frozen semen dilution in vitro survival time comparison
As can be seen from the table 2, the frozen and thawed bovine semen causes physiological and biochemical disorders to the plasma membrane of the sperm, which results in decreased sperm motility, damaged sperm membrane, broken DNA segments, leakage of intracellular enzymes, and ultimately affects insemination capability. Compared with the comparison groups 1-7, the experimental groups 1-3 can protect the ATP level of the frozen sperm to the maximum extent, effectively protect the acrosome of the sperm, improve the vitality of the frozen semen and prolong the survival time of the sperm.
2.2 CASA analysis
The sperm motility tracks were analyzed using a computer aided analyzer and the results are shown in table 3.
Wherein, the movement track of the sperm changes along with the change of the frozen semen, wherein VAP, VCL and BCF mainly represent the movement direction of the sperm. Wherein, the path velocity (VAP, m/s) refers to the ratio of the sperm head passing through a space mean parabola and the utilization time thereof, and the space mean parabola is a real parabola smoothed according to a certain arithmetic method. The mean path refers to the average distance of the sperm through space.
The tracking rate (VCL, m/s) refers to the ratio of the curve path of the head of the sperm through the true parabola and the time it takes.
The oscillation (whiplash) frequency (BCF, Hz) refers to the number of oscillations of the sperm tail per unit time, expressed as the number of intersections of the sperm head with the mean path per unit time.
TABLE 3 CASA test results
Table 3 shows the analysis of the sperm motility traces for survival time, and it can be seen that the frozen semen of the experimental groups 1-3 can still maintain higher motility speed and higher swing frequency after 18h and 24h of survival compared with the comparative groups 1-7.
Three, in vivo testing
3.1 first stage Artificial insemination conception test
Selecting 8 cattle from 4 pastures, wherein young cows and multiparous cows in the 8 cattle are respectively marked; the frozen diluents of the experimental group 1 and the conventional group frozen semen comparison groups 1-7 are labeled differently and sent to a pasture for carrying out an artificial insemination test, the statistical table is shown in table 4, and the conception rate of the artificial insemination period is shown in table 5.
Wherein the conventional group is obtained by purchasing frozen semen from cow center of Beijing first farm animal husbandry development Limited company.
Table 4 ranch providing frozen semen statistical table
Table 5 artificial insemination validation
As can be seen from table 5, the conception rates of the experimental group 1 were higher in both young cattle and multiparous cattle than in the conventional group.
3.2 popularization and application of frozen semen in the second stage
According to the test result of the first stage, in 2019, the semen of 10 bulls in a bulls station is completely produced by adopting the frozen semen diluent, 54827 doses of frozen semen are produced in a symbiotic manner until the semen reaches 2020.05.28 meshes, and the frozen semen is used comprehensively in 76 pastures, and the 45650 doses of frozen semen are used together. Statistics were performed on the 76 pasture conception rate data, and the results are shown in table 6.
The conventional group adopts the method of purchasing frozen semen of the cow center of Beijing first farm animal husbandry development Limited company.
Table 676 statistical analysis table for frozen semen data applied in pasture
| Estrus conception rate (young cattle) | Conception rate in love (through cattle) | |
| Experimental group 1 | 59.6％±4.9％* | 45.8％±3.1％* |
| General group | 57.7％±4.3％ | 42.7±2.0％ |
As can be seen from table 6, compared with the conventional group, the conception rate of the young cows is improved by 1.9%, and the conception rate of the adult cows is improved by 3.1%.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (5)
1. The frozen semen diluent for the dairy cow is characterized in that each 100mL of the frozen semen diluent comprises the following components:
20mL of yolk, 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 15 ten thousand IU of streptomycin, 75-80mg of cysteamine, 0.23-0.27g of reductive glutathione, 9.5-10.5mg of taurine, 3.5-5mg of disodium adenosine triphosphate, 2-2.5mL of refined white, 0.45-0.60mg of water-soluble laminarin and the balance of distilled water;
the pH value of the cow frozen semen diluent is 6.8-7.
2. The cow frozen semen diluent as claimed in claim 1, wherein the semen is cow semen.
3. A method of preparing a dilution of frozen cow semen according to claim 1, comprising: adding 2.32g of sodium citrate, 0.5g of fructose, 2.42g of Tris, 5.4g of glycerol, 75-80mg of cysteamine, 0.23-0.27g of reducing glutathione, 9.5-10.5mg of taurine, 3.5-5mg of disodium adenosine triphosphate and 0.45-0.60mg of water-soluble laminarin into distilled water, stirring uniformly, placing in a 75-80 ℃ water bath kettle for disinfection for 30min, cooling to normal temperature, then adding egg yolk, streptomycin and refined liquid, fixing the volume to 100mL by using distilled water, and adjusting the pH value to 6.8-7.
4. A method of semen freezing a frozen cow semen diluent as claimed in claim 1, comprising:
a) semen dilution
Adding the semen diluent into the semen to dilute the cow semen until the number of the semen in the solution reaches 2.4 multiplied by 106Per mL;
b) temperature reduction
Transferring the diluted semen into a thin tube, sealing, placing in a refrigerator at 4 deg.C, cooling, and balancing for 3-5 h;
c) freezing
Placing the balanced tubule semen stacking drawer in a full-automatic freezer, freezing, and finally placing in liquid nitrogen for preservation;
d) packaging frozen semen;
and (5) packaging and warehousing the frozen semen of the thin tube.
5. The method for freezing the semen of a cow by using the diluent of the frozen semen of the cow according to claim 4, wherein in the step c), the freezing comprises three stages, specifically:
in the first stage, the temperature of a partition plate before freezing is 23 ℃, and the temperature is reduced to 0-4 ℃ within 5 hours;
in the second stage, the freezing temperature is reduced to 0 to minus 5 ℃ at the cooling speed of 0.15 ℃/s, and the temperature is kept for 120 s;
and in the third stage, the freezing temperature is reduced to-5 to-130 ℃ at the cooling rate of 0.66 ℃/s, and is kept for 180 s.
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