CN116849207A - Low-temperature preservation diluent for semen of grouper with large body and use method and application thereof - Google Patents
Low-temperature preservation diluent for semen of grouper with large body and use method and application thereof Download PDFInfo
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Classifications
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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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a low-temperature preservation diluent for the semen of Epinephelus lanugreek, and a use method and application thereof. The diluent disclosed by the invention is prepared from the following raw materials: glucose, calcium chloride, potassium chloride, sodium bicarbonate, reduced glutathione, sodium dihydrogen phosphate and fetal bovine serum. Wherein, each liter of the diluent contains the following raw materials by mass: 0.4-0.5 g of glucose, 0.03-0.08 g of calcium chloride, 0.15-0.25 g of potassium chloride, 3-4 g of sodium chloride, 1.5-2.0 g of sodium bicarbonate, 1.5-2.5 g of reduced glutathione, 0.008-0.012 g of sodium dihydrogen phosphate, and 8-12% of the volume percentage of the fetal bovine serum. The diluent prepared by the invention can better maintain the in-vitro vigor of sperms, prolong the semen preservation time, and can meet the low-temperature preservation requirement of the grouper semen, in particular to the grouper semen with large blue body.
Description
Technical Field
The invention relates to the field of fish semen preservation, in particular to a low-temperature preservation diluent for the semen of Epinephelus lanuginosus, and a use method and application thereof.
Background
The Epinephelus lanuginosus belongs to the order of weever, the bass family, the Epinephelus subfamily and the Epinephelus genus, is commonly known as a macula, is one of large-sized species of the Epinephelus, and is widely distributed in India-Pacific areas, south China and other sea areas. The groupers with blue bodies grow fast and delicious in meat taste, and are important seawater economic fishes. The method has the advantages of high growth speed, but high seedling raising difficulty, low survival rate and small number of seedlings on the market, so the method is used as a male parent for hybridization with other groupers. As the research of the Epinephelus lansium is still in the starting stage, the related data are very few, and the system description of related species is not found in the literature at home and abroad. In actual production, the problems of asynchronous gonad development or insufficient sperm amount of male and female fishes are frequently encountered, so that a method for storing semen in a short period is needed, besides, after the semen is discharged from parent fishes, the body is lost from supplying nutrients, and the movement of the body can be stopped quickly only by the energy from the seminal plasma. The effective preservation means of the in-vitro sperms can create conditions for artificial insemination and even full artificial breeding.
Sperm has the ability to move, the needed energy comes from the nutrient substances of the protoplasm, but the sperm does not move when in a dense state, and for seawater fish fertilized in vitro, the sperm is activated after meeting seawater, and the sperm starts to move actively.
When fish sperms are stored in vitro, pollution is generally avoided when the sperms are obtained, and proper diluent can reduce the viscosity of the sperms, maintain homeostasis, supplement nutrition, reduce the influence of foreign matters, inhibit the movement of the sperms, alleviate the harm of metabolites and the like. Further determining proper dilution times of semen, and when the dilution times are too small, the concentration of semen is too high and sticky, and the significance of dilution is not too great, so that the activity is low; when the dilution factor is too large, the external morphological structure of the sperm is easy to change, and the activity is reduced. The product is placed at low temperature (0-4deg.C) or ultra-low temperature (liquid nitrogen or-80deg.C), and can inhibit metabolism of sperm, and prolong shelf life. In addition, low temperatures can also inhibit bacterial growth, which is also an important reason for extended shelf life. So far, reports or researches on low-temperature (0-4 ℃) preservation of the sperm of the groupers with blue bodies are not seen, only related ultralow-temperature preservation researches on the sperm are seen, but the method is suitable for cryopreservation and is complex in operation, liquid nitrogen and a liquid nitrogen tank are not normally reserved in a common farm, different personnel have different operations, the thawing time of the cryopreserved sperm is greatly influenced by the subjective effect, certain damage can be caused to the sperm by ultralow-temperature freezing, and the head plasma membrane of the sperm is broken, the middle section of the sperm falls off, cells die and the like. Meanwhile, a large amount of enzymes and proteins in sperms can enter seminal plasma due to the damage of the sperm membranes caused by freezing damage, so that the enzyme activity in sperms is reduced, the enzyme activity in seminal plasma is increased, and the condition of freezing damage can be increased along with the increase of freezing time.
At present, in actual production research, a diluent capable of effectively prolonging the external low-temperature preservation time of the semen of the grouper with large body is not available, which has great hindrance to related technicians who need to perform fine breed breeding and full-artificial breeding through artificial insemination, even if a freezing preservation technology exists, the short-term requirement that a general culturing farm without liquid nitrogen only needs to preserve for a plurality of hours or even days cannot be met, the operation is complex during freezing, the operation of different personnel has different, the thawing time of frozen sperm has great influence, the freezing preservation effect of sperm can be directly influenced, and even if some sperms are thawed, the inside and outside physiological and biochemical functions of the sperms are subjected to freezing damage, and the sperms do not have fertilization capacity.
The formula of the diluent is critical for the low-temperature preservation of semen, but no unified standard exists at present, and the general principle is as follows: the sea fish diluent should be isotonic or slightly hypotonic with the preserved semen; the buffer capacity is certain, and the pH can be maintained; contains certain nutrient substances to supply weak metabolism requirement of sperms at low temperature (0-4 ℃); comprises a substance for inhibiting sperm motility, such as K+ which inhibits sperm motility in salmon and trout species, and Na+ which reduces the inhibition; antibiotics can be added in proper amount to prevent a great deal of mold and the like from breeding in the preservation process; the medicines and vessels are strictly sterilized; it is ready for use.
The low temperature can reduce the metabolism of sperms, which is beneficial to prolonging the life of sperms, but too low temperature can generate ice crystals and damage sperms. Undiluted sperm can also be stored at low temperature (0-4 ℃) for a period of time without the metabolism stopping, but with a substantial decrease. During preservation, semen may produce a large amount of metabolic waste, some of which are toxic, and once the concentration is too high, sperm may be killed later. Lactic acid, carbon dioxide, etc. are also produced during metabolism, lowering semen pH. Besides, undiluted semen is easy to adhere and dry, collected fresh semen is high in density and sticky, the moisture in the seminal plasma is evaporated in the preservation process, surface sperms become increasingly dry, and inorganic bodies continuously supply nutrient substances required for maintaining metabolism, so that the preservation effect is greatly reduced. Therefore, a diluent is needed to meet the low-temperature preservation requirement of the grouper semen.
Disclosure of Invention
In view of the above, the invention provides a low-temperature preservation diluent for the semen of the groupers with large body, which can better maintain the in-vitro activity of the semen, prolong the preservation time and meet the low-temperature preservation requirement of the groupers with large body.
The technical scheme of the invention is realized as follows:
the low-temperature preservation diluent for the garrupa seminal fluid comprises the following raw materials: glucose, reduced glutathione, sodium dihydrogen phosphate and fetal bovine serum.
Further, the diluent also contains the following raw materials: calcium chloride, potassium chloride, sodium chloride and sodium bicarbonate.
Further, the diluent contains the following raw materials by mass per liter: 0.4-0.5 g of glucose, 0.03-0.08 g of calcium chloride, 0.15-0.25 g of potassium chloride, 3-4 g of sodium chloride, 1.5-2.0 g of sodium bicarbonate, 1.5-2.5 g of reduced glutathione, 0.008-0.012 g of sodium dihydrogen phosphate, and 8-12% of the volume percentage of the fetal bovine serum.
Further, the diluent contains the following raw materials by mass per liter: glucose 0.45g, calcium chloride 0.05g, potassium chloride 2g, sodium chloride 3.5g, sodium bicarbonate 1.69g, reduced glutathione 2g, sodium dihydrogen phosphate 0.01g, wherein the fetal bovine serum is 10v/v% of the volume percentage of the diluent.
Further, the preparation method of the preservation diluent comprises the following steps: respectively weighing glucose, calcium chloride, potassium chloride, sodium bicarbonate, reduced glutathione and sodium dihydrogen phosphate, and dissolving in water to obtain a base solution; before use, taking the fetal bovine serum, thawing, and then transferring to the base solution to prepare the preservation diluent.
The invention also provides a use method of the low-temperature preservation diluent of the large-body grouper semen, which comprises the steps of taking fresh semen and the preservation diluent according to the volume ratio of 1:1-99, mixing, and storing at low temperature. Further, the volume ratio is 1:9,1:29,1:49,1:69 or 1:99.
further, the volume ratio of the fresh semen to the preservation diluent is 1:49-69, and even more preferably a volume ratio of 1:49 or 1:69.
further, the low temperature storage temperature is 0-4 ℃.
Further, the low-temperature preservation time is less than or equal to 72 hours.
The diluent of any one of the invention is applied to preserving the grouper semen.
Further, the diluent is applied to preserving the semen of the grouper.
The invention also uses CASA technique to detect sperm motility. CASA technique: the technology is a new technology developed on the basis of a plurality of technologies and equipment including photomicrography, microscopic image analysis, fluorescence microscopy and the like, and is initially applied to male genitalia research, and is gradually applied to other mammal sperm motility detection along with the development and perfection of the technology. COSSON et al describe salmon sperm motility for the first time using stroboscope and video methods. As this technology matures, CASA technology has been widely used in recent years for the detection of sperm motility in fish and invertebrates. The CASA recognition technology mainly comprises DNA fluorescent recognition and gray scale recognition, adopts a microscopic video technology and an advanced image processing technology to comprehensively quantitatively analyze dynamic and static images of sperms, and can accurately measure parameters such as sperm movement rate (MOT), curve movement rate (VCL), linear movement rate (VSL), whipping frequency (BCF), side swing Amplitude (ALH) and the like.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention takes glucose, reduced glutathione, sodium dihydrogen phosphate, fetal bovine serum and the like as raw materials, and the ratio is optimized to prepare the preservation solution, thereby being applicable to the short-term low-temperature preservation requirement of the semen of the Epinephelus lanuginosa. The diluent prepared by the invention greatly prolongs the in-vitro preservation time of the semen of the Epinephelus lanugreek, can efficiently preserve the sperm of a high-quality male parent, provides technical support for artificial insemination of the Epinephelus lanugreek, and effectively meets the requirement of low-temperature (0-4 ℃) short-term preservation of the sperm during artificial fine variety breeding of the Epinephelus lanugreek.
(2) The diluent prepared by the invention can inhibit the activation of the sperm of the groupers and provide nutrition energy for the sperm; can reduce oxidative damage caused by metabolic products generated during the respiratory metabolism of the sperm of the groupers, and can also reduce low-temperature damage generated by the sperm of the groupers at low temperature. The diluent prepared by the invention can reduce the metabolism rate of sperms, reduce energy consumption and prolong the life of sperms. Wherein, the reduced glutathione in the formula of the invention is helpful for reducing oxidative damage caused by metabolic products generated during sperm respiratory metabolism; fetal Bovine Serum (FBS) not only helps to reduce oxidative damage caused by metabolites produced during respiratory metabolism of sperm, but also reduces low temperature damage produced by sperm at low temperatures.
(3) The diluted solution is added with calcium chloride, potassium chloride, sodium chloride and sodium bicarbonate, which is helpful for maintaining the pH value of the system, maintaining osmotic pressure and inhibiting sperm from being activated.
(4) The diluent prepared by the invention can better meet the low-temperature (0-4 ℃) preservation requirement of the garrupa semen, has no toxic effect on sperms, and effectively prolongs the sperm preservation time.
(5) The preparation and use of the diluent of the invention are simple and easy to operate, and after the low-temperature preservation solution is prepared in advance according to the programming requirement, the sperm can be preserved in the field by the ice box with the ice bag, thereby avoiding the damage to the sperm caused by the formation of ice crystals of the sperm below 0 ℃.
Drawings
Fig. 1 is a flowchart of the operation of embodiment 1 of the present invention.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The MS-222 used in the examples of the present invention was purchased from Shanghai Katine Biotech Co.
The reduced glutathione used in the embodiment of the invention is L-reduced glutathione, and the brand: microphone, goods number: G6268.
example 1
1. Preparing a diluent for preserving the semen of the grouper at low temperature:
1. weighing 0.45g of glucose, 0.05g of calcium chloride, 2g of potassium chloride, 3.5g of sodium chloride, 1.69g of sodium bicarbonate, 2g of reduced glutathione and 0.01g of sodium dihydrogen phosphate by an electronic balance, dissolving in ultrapure water at normal temperature, and fixing the volume to 0.9L to obtain a base solution, and preserving at 4 ℃ for later use with a preservation period of 1-3 days;
2. before use, taking out the fetal bovine serum at-20deg.C, completely dissolving at room temperature, transferring 0.1L into 0.9L of base solution, and mixing to obtain the low-temperature preservation solution.
2. Semen collection and dilution
1. Semen collection address is selected from yellow-flow Zhenjian village in Hainan Ledong Li Zu of Hainan province, 15 male fishes (Epinephelus lansium) which are subjected to nutrition enhancement, good growth and mature development of spermary are selected in breeding season, and semen is collected by means of manual extrusion. Before collection, the periphery of the colonial hole is wiped by a dry towel after the anesthesia of MS-222, so that the pollution samples such as moisture, urine and the like are prevented, the abdomen is lightly pressed to the colonial hole, after milky clean and pollution-free semen flows out, the liquid is immediately sucked by a pipetting gun with the measuring range of 200 mu L, and the liquid is lightly pumped into the bottom of a 5mL sterile EP tube without any liquid.
2. All semen collected is mixed for treatment, so that errors are reduced, inter-individual differences are eliminated, and the initial vitality is required to be more than 90%;
3. the semen and cryopreservation solutions were diluted 1:9,1:29,1:49,1:69,1:99 and placed in 5mL sterile EP tubes, which were experimental groups, with the total volume of each diluted semen being 2.1mL. The remaining fresh semen without any reagent added served as a control group.
4. All the EP pipes in the step 3 are wrapped with tinfoil paper and protected from light, so as to prevent light activation, then absorbent cotton is wrapped, the outermost gauze is tightly wrapped, and the wrapped absorbent cotton is placed in an ice box containing ice bags at the temperature of about 4 ℃.
3. Sperm motility assay
1. Detecting fresh essence activity: according to the fresh extract in the second step 3, 10min,2h,4h,6h,12h,24h,48h,72h and 96h after extracting the extract from the parent male fish are detected, and the specific operation steps of each detection are as follows: sucking 1 μL fresh semen into glass slide with 2.5 μL pipetting gun, adding 2 μL filtered natural seawater to activate semen, adjusting focal length until display screen imaging is clear, namely detecting sperm motility with sperm motility detection system (CASA) after final semen dilution 300 times, and mainly recording movement rate (MOT);
2. semen different dilution ratio detection:
1) When the dilution ratio v/v of the detection semen and the low-temperature preservation solution is 1:9 (equivalent to 10 times of dilution), the specific operation is as follows: sperm are taken out of the parent male fish, diluted according to the volume ratio of 1:9, stored at 4 ℃, and detected 10min,2h,4h,6h,12h,24h,48h,72h and 96h after the sperm are taken out of the parent male fish: adding 9 mu L of low-temperature preservation solution of the fine liquid of the Perch with leopard gill in a 200 mu L EP tube by using a 10 mu L pipette, sucking 1 mu L of diluted fine liquid with the dilution ratio of 1:9 according to the 2 in the second step by using a 2.5 mu L pipette, and lightly blowing and uniformly mixing; sucking 1 mu L of mixed solution on a glass slide, adjusting the focal length until the imaging of a display screen is clear, adding 2 mu L of filtered natural seawater to activate sperms, namely detecting the sperm motility by using a sperm motility detection system (CASA) after the final semen is diluted 300 times, and mainly recording the motility rate (MOT);
2) When the dilution ratio v/v of the detection semen and the low-temperature preservation solution is 1:29 (which is equivalent to 30 times of dilution), the specific operation is as follows: sperm are taken out of the parent male fish, diluted according to the proportion of 1:29, stored at 4 ℃, and detected 10min,2h,4h,6h,12h,24h,48h,72h and 96h after the sperm are taken out of the parent male fish: adding 2.8 mu L of the low-temperature preservation solution of the garrupa seminal fluid into a 200 mu L EP tube by using a 2.5 mu L liquid-transferring gun, sucking 1.2 mu L of diluted seminal fluid with the dilution ratio of 1:29 according to 3 in the second step by using the 2.5 mu L liquid-transferring gun, and lightly blowing and uniformly mixing; sucking 1 mu L of mixed solution on a glass slide, adding 2 mu L of filtered natural seawater to activate sperms, adjusting the focal length until the imaging of a display screen is clear, namely detecting the sperm motility by using a sperm motility detection system (CASA) after the final semen is diluted 300 times, and mainly recording the motility rate (MOT);
3) When the dilution ratio v/v of the detection semen and the low-temperature preservation solution is 1:49 (which is equivalent to 50 times of dilution), the specific operation is as follows: sperm are taken out of the parent male fish, diluted according to the volume ratio of 1:49, stored at 4 ℃, and detected 10min,2h,4h,6h,12h,24h,48h,72h and 96h after being taken out of the parent male fish: adding 1 mu L of the low-temperature preservation solution of the garrupa seminal fluid into a 200 mu L EP tube by using a 2.5 mu L pipette, sucking 1 mu L of diluted seminal fluid with the dilution ratio of 1:49 according to 3 in the second step by using the 2.5 mu L pipette, and lightly blowing and uniformly mixing; sucking 1 mu L of mixed solution on a glass slide, adding 2 mu L of filtered natural seawater to activate sperms, adjusting the focal length until the imaging of a display screen is clear, namely detecting the sperm motility by using a sperm motility detection system (CASA) after the final semen is diluted 300 times, and mainly recording the motility rate (MOT);
4) When the dilution ratio of the detection semen to the low-temperature preservation solution is 1:69 (which is equivalent to 70 times of dilution), the specific operation is as follows: sperm are taken out of the parent male fish, diluted according to the volume ratio of 1:69, stored at 4 ℃, and detected 10min,2h,4h,6h,12h,24h,48h,72h and 96h after being taken out of the parent male fish: adding 0.6 mu L of the low-temperature preservation solution of the garrupa seminal fluid into a 200 mu L EP tube by using a 2.5 mu L liquid-transferring gun, sucking 1.4 mu L of diluted seminal fluid with the dilution ratio of 1:69 according to 3 in the second step by using the 2.5 mu L liquid-transferring gun, and lightly blowing and uniformly mixing; sucking 1 mu L of mixed solution on a glass slide, adding 2 mu L of filtered natural seawater to activate sperms, adjusting the focal length until the imaging of a display screen is clear, namely detecting the sperm motility by using a sperm motility detection system (CASA) after the final semen is diluted 300 times, and mainly recording the motility rate (MOT);
5) When the dilution ratio v/v of the detected semen and the low-temperature preservation solution is 1:99 (which is equivalent to 100 times of dilution), the specific operation is as follows: sperm are taken out of the parent male fish, diluted according to the volume ratio of 1:99, stored at 4 ℃, and detected 10min,2h,4h,6h,12h,24h,48h,72h and 96h after the sperm are taken out of the parent male fish: sucking 1 mu L of diluted semen with the dilution ratio of 1:99 according to the second step by using a 2.5 mu L pipetting gun, adding 2 mu L of filtered natural seawater to activate the semen, adjusting the focal length until the image of the display screen is clear, namely detecting the sperm motility by using a sperm motility detection system (CASA) after the final semen is diluted 300 times, and mainly recording the movement rate (MOT);
after the motility of the sperms is detected according to the method described in the third step, the obtained data are shown in a table (1), and it can be known that the motility of the sperms is highest at the dilution ratio of 1:49 and 1:69, and is 47.56% and 44.53% respectively at 24h, which is superior to the motility of fresh sperms, and the motility of the sperms exceeds 10% at 72h, which indicates that the highest preservation time of the sperms is 3d, namely 72h at the dilution of 50 times and 70 times, and the motility of all experimental groups and control groups is 0, and the total death can be understood.
Table (1) sperm motility at different dilution ratios for Epinephelus rupestris with blue body and different storage times
Note that: "-" indicates no activation, and the movement rate is 0, namely death.
4. Artificial insemination
According to the result obtained in the third step, the preservation time of the grouper semen and the low-temperature preservation solution with the dilution ratio of 1:49 can reach about 72 hours, and 47.56 percent of sperms can be still activated before 24 hours. Therefore, in artificial insemination experiments, semen was diluted 50-fold as the experimental group and fresh semen as the control group. The wet fertilization is adopted, after 50 mu L of diluted semen is activated by seawater in an experimental group, 2mL of ovum is immediately poured into the culture dish containing the activated semen for a plurality of minutes, the mixture is continuously stirred and then is transferred into clean seawater at 28 ℃ for hatching, and the 50 mu L of diluted semen is replaced by 1 mu L of fresh semen in a control group. Fertilize with ovum after 2h,6h,12h,24h after storage, and count fertilization rate and hatching rate.
Fertilization rate= (number of embryos developed to 4-16 cell stage/total number of eggs) ×100%;
hatchability = (number of initially hatched fish/number of fertilized embryos) ×100%.
The eggs extruded from the parent female fish are good in development, spherical, homogeneous and transparent in appearance, and most of the eggs float on the surface of seawater.
According to the artificial insemination procedure, experimental data in table (2) are obtained, and according to the results, the fertilization ability of the diluted semen is slightly better than that of fresh semen when the semen is stored for 2 hours at low temperature; the fertilization rate of the groupers with large body is rapidly reduced when the fresh sperm of the groupers with large body is refined for 6 hours; the diluted semen still has higher fertilization rate and hatching rate of 70.03 percent and 64.78 percent respectively when being stored for 24 hours at low temperature, and still can meet the actual production requirement.
TABLE (2 fertilization rates and hatchability after artificial insemination at different time periods and different dilution ratios)
Note that: "-" indicates that the fertility rate or hatching rate is 0.
Example 2
The amounts of the respective raw materials were adjusted based on example 1. Preparing a diluent for preserving the semen of the grouper at low temperature:
weighing 0.40g of glucose, 0.05g of calcium chloride, 1.5g of potassium chloride, 3.7g of sodium chloride, 1.54g of sodium bicarbonate, 2.5g of reduced glutathione and 0.01g of sodium dihydrogen phosphate by an electronic balance, dissolving in ultrapure water at normal temperature, fixing the volume to 0.9L to obtain a base solution, and preserving at 4 ℃ for later use; before use, taking out the fetal bovine serum at-20deg.C, completely dissolving at room temperature, transferring 0.1L into 0.9L of base solution, and mixing to obtain the low-temperature preservation solution. The low-temperature preservation solution can meet the short-term preservation effect requirement of the grouper semen.
Example 3
The amounts of the respective raw materials were adjusted based on example 1. Preparing a diluent for preserving the semen of the grouper at low temperature:
weighing 0.50g of glucose, 0.04g of calcium chloride, 2.5g of potassium chloride, 3.4g of sodium chloride, 1.75g of sodium bicarbonate, 1.5g of reduced glutathione and 0.01g of sodium dihydrogen phosphate by an electronic balance, dissolving in ultrapure water at normal temperature, fixing the volume to 0.9L to obtain a base solution, and preserving at 4 ℃ for later use; before use, taking out the fetal bovine serum at-20deg.C, completely dissolving at room temperature, transferring 0.1L into 0.9L of base solution, and mixing to obtain the low-temperature preservation solution. The low-temperature preservation solution can meet the short-term preservation effect requirement of the grouper semen.
Example 4
Based on example 1, the reduction feed was adjusted to be free of calcium chloride, potassium chloride, sodium chloride and sodium bicarbonate. Preparing a diluent for preserving the semen of the grouper at low temperature: weighing 0.45g of glucose, 2g of reduced glutathione and 0.01g of sodium dihydrogen phosphate by an electronic balance, dissolving in ultrapure water at normal temperature, and keeping the volume to 0.9L to obtain a base solution, and preserving at 4 ℃ for later use; before use, taking out the fetal bovine serum at-20deg.C, completely dissolving at room temperature, transferring 0.1L into 0.9L of base solution, and mixing to obtain the low-temperature preservation solution. Compared with the embodiment 1, the low-temperature preservation solution has a reduced preservation effect on the semen of the Epinephelus lancifolius, but can meet the requirement of short-term preservation effect on the semen of the Epinephelus lancifolius.
Comparative example 1
Based on example 1, reduced glutathione was replaced with a soy polypeptide (brand name of Huayuan Cheng Tai). Preparing a diluent for preserving the semen of the grouper at low temperature: weighing 0.45g of glucose, 0.05g of calcium chloride, 2g of potassium chloride, 3.5g of sodium chloride, 1.69g of sodium bicarbonate, 2g of soybean polypeptide and 0.01g of sodium dihydrogen phosphate by an electronic balance, dissolving in ultrapure water at normal temperature, fixing the volume to 0.9L to obtain a base solution, and preserving at 4 ℃ for later use; before use, taking out the fetal bovine serum at-20deg.C, completely dissolving at room temperature, transferring 0.1L into base solution 0.9L, and mixing to obtain preservation solution.
Fresh semen (activity greater than 90%) and preservation solution were diluted 1:9,1:29,1:49,1:69,1:99 and placed into a 5mL sterile EP tube, and set as comparative groups 1-5 in sequence, with the total volume of each diluted semen being 2.1mL. The results showed that the sperm motility was less than 20% after 24h storage for each group. The effect of preserving the seminal fluid of the groupers with blue bodies is obviously lower than that of the examples.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The low-temperature preservation diluent for the garrupa seminal fluid is characterized by comprising the following raw materials: glucose, reduced glutathione, sodium dihydrogen phosphate and fetal bovine serum.
2. The low-temperature preservation diluent for the semen of the grouper body according to claim 1, which is characterized by further comprising the following raw materials: calcium chloride, potassium chloride, sodium chloride and sodium bicarbonate.
3. The low-temperature preservation diluent for the semen of the grouper body according to claim 2, wherein each liter of the diluent comprises the following raw materials by mass: 0.4-0.5 g of glucose, 0.03-0.08 g of calcium chloride, 0.15-0.25 g of potassium chloride, 3-4 g of sodium chloride, 1.5-2.0 g of sodium bicarbonate, 1.5-2.5 g of reduced glutathione, 0.008-0.012 g of sodium dihydrogen phosphate, and 8-12% of the volume percentage of the fetal bovine serum.
4. The low-temperature preservation diluent for the semen of the grouper body according to claim 2, wherein each liter of the diluent comprises the following raw materials by mass: glucose 0.45g, calcium chloride 0.05g, potassium chloride 2g, sodium chloride 3.5g, sodium bicarbonate 1.69g, reduced glutathione 2g, sodium dihydrogen phosphate 0.01g, wherein the fetal bovine serum is 10v/v% of the volume percentage of the diluent.
5. The low-temperature preservation diluent for the semen of the grouper according to any one of claims 1 to 4, wherein the preparation method of the preservation diluent comprises the following steps: respectively weighing glucose, calcium chloride, potassium chloride, sodium bicarbonate, reduced glutathione and sodium dihydrogen phosphate, and dissolving in water to obtain a base solution; before use, taking the fetal bovine serum, thawing, and then transferring to the base solution to prepare the preservation diluent.
6. The method for using the low-temperature preservation diluent for the semen of the grouper body according to claim 1, wherein the volume ratio of the fresh semen to the preservation diluent is 1:1-99, mixing, and storing at low temperature.
7. The method for using the low-temperature preservation diluent for the semen of the grouper according to claim 5, wherein the volume ratio of the fresh semen to the preservation diluent is 1: 49-69.
8. The method for using the low-temperature preservation diluent of the semen of the grouper with blue body according to claim 1, wherein the low-temperature preservation temperature is 0-4 ℃.
9. Use of the diluent according to claims 1-5 for preserving garrupa semen.
10. Use of the diluent according to claim 9 for preserving the semen of grouper.
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