CN116267896B - Application of squalene in improving survival rate of bee sperms and semen preservation method - Google Patents
Application of squalene in improving survival rate of bee sperms and semen preservation method Download PDFInfo
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- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 title claims abstract description 41
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 title claims abstract description 41
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 title claims abstract description 41
- 229940031439 squalene Drugs 0.000 title claims abstract description 41
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 title claims abstract description 41
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- 238000004321 preservation Methods 0.000 title abstract description 20
- 239000003761 preservation solution Substances 0.000 claims abstract description 24
- 238000003879 sperm preservation Methods 0.000 claims abstract description 24
- 239000007853 buffer solution Substances 0.000 claims abstract description 18
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 241000257303 Hymenoptera Species 0.000 claims description 8
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- 238000002156 mixing Methods 0.000 claims description 8
- 239000001103 potassium chloride Substances 0.000 claims description 8
- 235000011164 potassium chloride Nutrition 0.000 claims description 8
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000019100 sperm motility Effects 0.000 description 7
- 230000006378 damage Effects 0.000 description 6
- 230000035899 viability Effects 0.000 description 6
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- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- 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
-
- 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
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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 discloses an application of squalene in improving the survival rate of bee sperms and a semen preservation method. The invention uses the squalene in the protection process of the bee sperms, fully utilizes the excellent antioxidant capacity of the squalene in the sperm preservation process, and remarkably improves the activity and survival rate of the sperms; when 0.5mol/L squalene is added into the sperm preservation solution for sperm preservation, the in-vitro survival rate of the sperm is 27% higher than that of the sperm preservation solution without squalene when the sperm is preserved for 36 hours. The preservation method has the advantages of simple process, easy operation and lower cost; in the semen preservation solution, squalene is easy to obtain, the preparation method of the buffer solution is simple, the cost is further reduced, and the semen preservation solution has good application prospect.
Description
Technical Field
The invention belongs to the technical field of sperm preservation, and particularly relates to application of squalene in improving the sperm motility and survival rate of bees and a sperm preservation method.
Background
Bees are an important economic insect, and at the same time, bees are also an important ecological insect which produces a high ecological value for pollination of crops. However, along with factors such as ecological destruction and large-area pesticide use, bee germplasm resources are destroyed, and people pay more attention to protecting the bee germplasm resources.
At present, bee sperms are usually preserved for a long time by adopting a low-temperature refrigeration mode, and a common buffer solution for preservation mainly comprises an improved basic auxiliary solution (sodium citrate 3.6g and NaHCO) 3 0.32g, KCl 0.06g, glucose 0.5g, double distilled water 100 ml), ringer's solution (NaCl 0.85g, KCl 0.025g, caCl) 2 0.03g, 0.5g of glucose, 100ml of double distilled water), jacquard solution (NaCl 0.7g, KCl 0.025g, glucose 0.1g, 100ml of double distilled water), rockwell solution (NaCl 0.85g, KCl 0.025g, caCl) 2 0.01g、NaHCO 3 0.01g, 0.1g of glucose, 100ml of double distilled water, and modified physiological saline (NaCl 0.85g, 0.5g of glucose, 100ml of double distilled water). However, damage to sperm, such as oxidative damage, mitochondrial damage, and plasma membrane damage, often occurs during in vitro preservation; wherein the primary injury is caused by oxidative stress. Such as Reactive Oxygen Species (ROS), are substances necessary for maintaining normal function of sperm, but excessive ROS accumulate harmful substances, resulting in impaired sperm physiology and motility, and reduced motility. Therefore, reducing the damage of sperms caused by oxidative stress is of great importance for in vitro preservation of sperms. Often, cryoprotectants such as glycerol, DMSO, etc. are added to the buffer to maximize protection of semen from low temperatures and other ROS. However, in 2012 Wegener J. Et al, research on the influence of cryoprotectants on queen bee reproductive health shows that cryoprotectants can reduce the migration quantity of sperm to queen bee fertilized capsules, greatly reducing insemination success rate.
Therefore, there is an urgent need to find a more biological, low toxicity, reliable biological cryoprotectant to ensure sperm motility. The method is also a great thrust for preservation and inheritance of bee germplasm resources, and has very important practical economic value.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to use squalene as a biological cryoprotectant for preserving bee sperm and improving the activity and survival rate of bee sperm. And further provides a preservation method of the bee semen, which solves the technical problem of sperm motility reduction caused by oxidative damage in the in-vitro preservation process of the sperm in the prior art.
In order to solve the problems, the technical scheme of the invention is as follows:
use of squalene for increasing sperm survival rate of bees.
A preservation method of bee semen, comprising the following steps:
s1, mixing a bee sperm stock solution with a buffer solution to obtain a sperm mixed solution; wherein, the volume percentage of the sperm stock solution and the buffer solution is 1:1, a step of;
s2, adding squalene into the sperm mixed solution, uniformly mixing to obtain sperm preservation solution, sealing and preserving at 4 ℃; wherein the concentration of squalene in the sperm preservation solution is 0.5mol/L-1mol/L;
preferably, the concentration of squalene in the sperm preservation solution is 0.5mol/L.
Further, the bee sperm stock solution is prepared from bee semen and distilled water according to the volume percentage of 1:1, and mixing.
Further, the buffer solution is prepared from the following components in percentage by mass: trisodium citrate dihydrate 3.6%, potassium chloride 0.06%, sodium bicarbonate 0.32%, glucose 0.5%, and distilled water in balance.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention firstly proposes that the squalene is used for improving the vitality and the survival rate of the sperms in the preservation process, and the squalene is used for protecting the sperms of bees, so that the excellent antioxidant capacity of the squalene in the preservation process of the sperms is fully utilized, and the vitality and the survival rate of the sperms are obviously improved; when 0.5mol/L squalene is added into the sperm preservation solution for sperm preservation, the in-vitro survival rate of the sperm is 27% higher than that of the sperm preservation solution without squalene when the sperm is preserved for 36 hours.
2. According to the preservation method of the bee semen, the preservation of the sperms can be completed by sequentially adding the buffer solution and squalene into the sperm stock solution and refrigerating at a low temperature. The process is simple, the operation is easy, and the cost is low; in the semen preservation solution, squalene is easy to obtain, the preparation method of the buffer solution is simple, the cost is further reduced, and the semen preservation solution has good application prospect.
Drawings
FIG. 1 is a plot of sperm viability in vitro versus time for examples 1, 2 and comparative example 1 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples.
Numerical ranges in this disclosure are understood to also specifically disclose each intermediate value between the upper and lower limits of the ranges. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The experimental methods used in the present invention are conventional methods unless otherwise specified.
The materials, reagents and the like used in the present invention can be synthesized by a method of purchase or known method unless otherwise specified.
In the quantitative test of the invention, three repeated experiments are set, and the results are averaged.
First, the invention creatively proposes the application of squalene in improving the sperm motility and survival rate of bees.
Wherein, squalene is identified from the seminal vesicle of drone by GC-MS technology, and is a nontoxic enhancer with anti-aging and anticancer effects. It is proved to have the biological activity effects of improving anoxia tolerance, inhibiting microorganism growth, resisting bacteria, diminishing inflammation, regulating cholesterol metabolism, etc. in other materials such as microorganism, plant seed, microalgae, shark liver and sebum. The invention uses squalene for preserving bee semen, and aims to provide a novel protective substance for semen storage, reduce semen activity injury caused by a protective agent, and avoid affecting reproductive health of queen bee.
Furthermore, the invention also provides a method for preserving semen by using squalene.
1. Examples
Example 1
A preservation method of bee semen, comprising the following steps:
(1) Preparing semen preservation solution
Preparing a buffer solution: weighing 3.6g of trisodium citrate dihydrate, 0.06g of potassium chloride, 0.32g of sodium bicarbonate and 0.5g of glucose, dissolving with distilled water, transferring into a 100mL volumetric flask, and fixing the volume to 100mL with distilled water; the pH value of the buffer solution is 8.0, is slightly alkaline, is similar to the pH value of the internal environment of the royal jelly capsule, and is more beneficial to preserving semen. Wherein, the trisodium citrate dihydrate has good pH adjustment and buffering performance, the potassium chloride and the sodium bicarbonate play a role in balancing the pH of the buffer solution, and the glucose is a main substance for supplying energy.
Preparing squalene solution: squalene from Shanghai Meilin Biochemical technologies Co., ltd was diluted to 1mol/L and 2mol/L with distilled water.
(2) Extraction of semen
Selecting sexually mature drones, pressing the abdomen to promote eversion of male stems, collecting semen by a capillary tube, transferring the collected semen into a sterile PCR tube, adding distilled water for dilution to obtain a sperm stock solution, wherein the volume ratio of the semen to the distilled water is 1:1.
(3) Preserving semen
The buffer solution obtained in the step (1) and the sperm stock solution obtained in the step (2) are mixed according to the volume ratio of 1:1, adding the squalene solution (1 mol/L or 2 mol/L) obtained in the step (1) after mixing, uniformly mixing to obtain a sperm preservation solution, wrapping a container for containing the sperm preservation solution by using tin foil paper, and refrigerating at 4 ℃. Wherein the concentration of squalene in the sperm preservation solution is 0.5mol/L.
Example 2
The preservation method of bee semen is the same as in example 1, except that the concentration of squalene in the sperm preservation solution is 1mol/L.
Comparative example 1
The preservation method of bee semen is the same as in example 1, except that squalene is not added to the sperm preservation solution.
Comparative examples 2 to 5
The preservation method of bee semen is the same as that of comparative example 1, except that the buffer composition is different.
2. Sperm motility assay
Sperm motility was measured after 0h, 12h, 24h, and 36h of storage of sperm stored in the sperm storage solutions obtained in examples 1 and 2 and comparative example 1, respectively. Sperm viability is shown in table 1 (also available from figure 1).
TABLE 1 determination of sperm viability at different time periods
From Table 1, in the in vitro 0-36h sperm storage experiment, squalene with different concentrations has certain protection effect on sperm motility; wherein, the storage effect of the sperm is best when the concentration of squalene in the sperm preservation solution is 0.5mol/L. From a comparison of example 1 and comparative example 1, it is evident that the addition of squalene significantly increases the survival rate of sperm, especially 36h sperm survival rate significantly different (p < 0.05) from comparative example 1. From a comparison of example 2 and comparative example 1, however, it is understood that at 24h sperm survival rate example 2< comparative example 1, and at 36h sperm survival rate example 2> comparative example 1, the inventors analyzed that squalene plays a greater role in the second half of sperm storage.
The detection method comprises the following steps: dye solutions of corresponding working concentrations were prepared according to kit instructions prior to each staining using Hoechst 33342 and PI double staining methods, and the dye solutions were added to sperm preservation solutions as described in examples and comparative examples, and the resulting solutions were then dripped onto a cytometer plate for viable and dead cell counting using the cytometer plate. The nuclei of living cells were stained blue with Hoechst 33342 reagent, while the nuclei of dead cells were stained red with PI reagent. Sperm count and viability were obtained by counting the number of blue and red nuclei.
Specific: before the experiment starts, respectively adding Hoechst 33342 dye liquor into sperm preservation solutions obtained in the examples and the comparative examples, centrifuging for 4-5s, ensuring that the dye liquor and semen are fully mixed, and incubating for 30min at 37 ℃ in a dark place; immediately after the addition of PI dye, incubation was carried out at 37℃for 10min in the dark. And finally, dripping the obtained solution on a blood cell counting plate to ensure that the solution covers the whole counting area, photographing the sperms in the upper left, lower left, upper right, lower right and middle area of the middle counting area under the fields of white light and 200 times of fluorescence, and counting. Calculation according to the formula for 25x16 cytometer: t= (l1+l2+r1+r2+m)/80×400×10000×d, where T is the total count, L1 is the number of upper left regions, L2 is the number of lower left regions, R1 is the number of upper right regions, R2 is the number of lower right regions, M is the number of intermediate regions, and D is the dilution ratio. The number of viable cells was defined in our experimental results as sperm motility (i.e., the number of blue nuclei), and the total number was the sum of the number of sperm in all fields of view (i.e., the sum of the number of blue nuclei and red nuclei). All dye solutions were purchased from Shanghai Meilin Biochemical technologies Co.
Sperm motility was measured after the sperm preservation solutions obtained in comparative examples 1 to 5 were preserved for 10min, 4h, 12h, 20h, and 28h, respectively. Sperm viability is shown in table 2.
Table 2 comparative sperm viability statistics
| Comparative example | 10min | 4h | 12h | 20h | 28h |
| Comparative example 1 | 82.7％ | 68.53％ | 55.63％ | 33.6％ | 28％ |
| Comparative example 2 | 78.73％ | 66.13％ | 39.36％ | 0 | 0 |
| Comparative example 3 | 73.16％ | 68.2％ | 43.36％ | 6.2％ | 0 |
| Comparative example 4 | 83.5％ | 67.96％ | 49.35％ | 21.26％ | 0 |
| Comparative example 5 | 70.66％ | 62.16％ | 38.93％ | 0 | 0 |
As can be seen from the comparative example, the buffer solution of the invention is used for preserving the bee semen, and the sperm survival rate is obviously superior to other buffer solutions. And in combination with the sperm preservation solution added with squalene, the preserved bee sperm has a survival rate remarkably superior to that of comparative example 1. Therefore, squalene has great significance for improving the survival rate of the sperm of bees, and meanwhile, compared with other buffers, the buffer solution selected by the invention has the effect of obviously improving the survival rate of the sperm.
In addition, the preservation method of the bee semen has the advantages of simple process, easy operation and lower cost; and squalene is easy to obtain, the preparation method of the buffer solution is simpler, the cost is further reduced, and the method has good application prospect.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the technical solution, and those skilled in the art should understand that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the present invention, and all such modifications and equivalents are included in the scope of the claims.
Claims (3)
1. Use of squalene for increasing sperm survival rate of bees.
2. A method for preserving honeybee semen, comprising the steps of:
s1, mixing a bee sperm stock solution with a buffer solution to obtain a sperm mixed solution; wherein, the volume percentage of the sperm stock solution and the buffer solution is 1:1, a step of; the buffer solution is prepared from the following components in percentage by mass: trisodium citrate dihydrate 3.6%, potassium chloride 0.06%, sodium bicarbonate 0.32%, glucose 0.5%, and distilled water in balance;
s2, adding squalene into the sperm mixed solution, uniformly mixing to obtain sperm preservation solution, sealing and preserving at 4 ℃; wherein the concentration of squalene in the sperm preservation solution is 0.5mol/L.
3. The method for preserving honeybee semen according to claim 2, wherein the honeybee sperm stock solution is prepared from honeybee semen and distilled water in a volume percentage of 1:1, and mixing.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310236946.7A CN116267896B (en) | 2023-03-13 | 2023-03-13 | Application of squalene in improving survival rate of bee sperms and semen preservation method |
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