CN117063919B - Application of pyrroloquinoline quinone in improving semen freezing effect - Google Patents
Application of pyrroloquinoline quinone in improving semen freezing effect 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
- A01N1/0226—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
-
- 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 provides application of pyrroloquinoline quinone in improving semen freezing effect. The method for improving the semen freezing effect comprises the step of adding semen into a freezing preservation solution containing pyrroloquinoline quinone for freezing preservation. The method can effectively reduce the structural damage and oxidation damage of the sperms in the freezing process, thereby improving the activity and oxidation resistance of the sperms after freezing preservation and effectively improving the quality of frozen semen.
Description
Technical Field
The invention belongs to the technical field of animal husbandry production, and particularly relates to application of pyrroloquinoline quinone in improving semen freezing effect.
Background
Sperm cells produce large amounts of reactive oxygen species (Reactive Oxygen Species, ROS) during cryopreservation at ultra-low temperatures, and at normal temperatures, sperm cells have a set of antioxidant systems that allow ROS to be at a normally low concentration, which is important to maintain tissue integrity. However, under the condition of freezing and preserving at ultralow temperature (-196 ℃ to-79 ℃), the enzyme activity related to oxidation resistance in the cells is inhibited, so that the ROS content in the cells is continuously increased. These ROS react with lipids on the cell membrane, causing irreversible damage to sperm cell membranes, mitochondria and DNA, greatly reducing sperm quality.
Pyrroloquinoline quinone (pyrroloquinoline quineone, PQQ) is a novel redox cofactor different from nicotinamide nucleotide and flavin nucleotide, widely existing in microorganisms, animals, plants and human bodies, and has a promoting effect on the growth and development of animals and plants. Numerous studies have shown that the lack of PQQ will cause serious health problems in mammals, such as growth retardation and immune system injury. There are also studies showing that PQQ has a good protective effect on both brain and cardiovascular disease in mice and can inhibit the progression of diabetes. The role of PQQ in reproduction has also been reported in small numbers, but its effect on female reproduction has been studied. The results of Steinberg F et al show that lack of PQQ in the diet can significantly reduce fertility in female mice or rats, primarily as a result of reduced conception rate and reduced litter size.
The frozen semen artificial insemination technology plays a vital role in the rapid propagation of seed industry, and the quality of frozen semen directly relates to the conception rate after insemination. How to effectively improve the frozen quality of the bovine semen, and the frozen preservation solution plays a key role. Therefore, improving the freezing effect of bovine semen by optimizing the formula of the freezing preservation solution is a technical problem to be solved.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of pyrroloquinoline quinone in semen cryopreservation on one hand and a method for improving semen cryopreservation effect on the other hand.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the application of pyrroloquinoline quinone in semen cryopreservation is used for improving the semen cryopreservation effect. Further, pyrroloquinoline quinone is used to reduce structural and oxidative damage to sperm during freezing, and pyrroloquinoline quinone is added prior to semen freezing.
In the application, preferably, the freezing preservation is carried out in a freezing preservation solution containing pyrroloquinoline quinone, and the concentration of the pyrroloquinoline quinone in the freezing preservation solution is 50-150 mu mol/L; more preferably 100. Mu. Mol/L.
The applications described above are one or more of the following:
pyrroloquinoline quinone improves the motility of sperm after semen is frozen and preserved;
pyrroloquinoline quinone improves the plasma membrane integrity of sperm after semen is frozen and preserved;
pyrroloquinoline quinone improves the acrosome integrity of sperm after semen is frozen and preserved; pyrroloquinoline quinone reduces the ROS level of sperm after cryopreservation of semen;
pyrroloquinoline quinone reduces the MDA level of sperm after cryopreservation of semen;
pyrroloquinoline quinone increases ATP levels in sperm after cryopreservation.
For the above application, preferably, the semen is bovine semen, murine semen, porcine semen, human semen, sheep semen.
Application of pyrroloquinoline quinone in preparing semen cryopreservation liquid is provided.
The invention also provides a semen cryopreservation solution which contains pyrroloquinoline quinone.
As for the semen cryopreservation liquid, preferably, the concentration of pyrroloquinoline quinone in the semen cryopreservation liquid is 50-150 mu mol/L.
Preferably, the semen cryopreservation liquid is an aqueous solution containing 10-20% of egg yolk, 4-8% of fructose and 4-8% of sodium citrate by mass percent;
further preferably, the concentration of the pyrroloquinoline quinone in the semen cryopreservation liquid is 100 mu mol/L.
A method for improving semen cryopreservation effect comprises adding semen into cryopreservation solution containing pyrroloquinoline quinone for cryopreservation.
In the method, preferably, the concentration of the pyrroloquinoline quinone is 50-150 mu mol/L, and the semen comprises but is not limited to cow semen, mouse semen, pig semen, human semen and sheep semen.
Further, the concentration of pyrroloquinoline quinone is preferably 100 [ mu ] mol/L.
In the above method, the cryopreservation liquid is preferably an aqueous solution containing 10 to 20% by mass of egg yolk, 4 to 8% by mass of fructose and 4 to 8% by mass of sodium citrate, and most preferably the cryopreservation liquid is an aqueous solution containing 15% by mass of egg yolk, 6% by mass of fructose and 6% by mass of sodium citrate.
The invention has the beneficial effects that:
the method for improving the semen freezing preservation effect can effectively reduce the structural damage and the oxidation damage of the sperms in the freezing process, thereby improving the vitality and the oxidation resistance of the sperms after freezing preservation, and further effectively improving the quality of frozen semen.
The invention also provides a new application of the pyrroloquinoline quinone, and the pyrroloquinoline quinone is added into the semen cryopreservation liquid, so that the quality of the cryopreserved semen can be effectively improved, and a certain technical support is provided for the preservation of the bovine semen.
Compared with GSH used in the prior art as a protective agent, the PQQ is far lower in dosage than GSH (which is an order of magnitude lower than GSH), and the effect is remarkably improved.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
Unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, all reagents used in the examples are analytically pure or of the above specification.
Example 1
1. Semen collection: 3 breeding bull with healthy constitution, moderate size and vigorous sexual desire are selected at suburb breeding bull stations, and semen is collected by adopting a pseudo-vagina method. The volume of normal semen is about 5ml, and the semen has a milky white or beige color, and the light shaking semen forms a cloud mist, which indicates that the semen quality is good; if the semen volume is not consistent with the expected serious or the semen color is transparent, egg yellow and the like, the semen quality is not qualified, and the semen of the cow needs to be replaced again.
2. Semen dilution and treatment
The raw semen was kept in a refrigerator at 17℃for 30min and then stored in an incubator with equal volume dilution of EMCARE Holding Solution dilution (available from ICPBio, reduction Co.) and semen. During transportation, semen is gently shaken every 30min, so that metabolism and energy consumption of the semen are inhibited by reducing the temperature, meanwhile, the influence of the difference of the original semen and the diluent on the semen is prevented, the semen is prevented from striking, and a semen sample is transported to a laboratory within 2 h.
Semen and EMCARE Holding Solution diluent are mixed according to the volume ratio of 1:2, diluting, after diluting, taking 1 μl semen and 200 μl semen density detection liquid, uniformly mixing, checking the sperm density by a sperm density detector, sucking 4 μl semen to prepare a slide specimen, observing and counting under a 400-time microscope, and repeating for at least 3 fields of view for three times for each sample. If the activity rate is more than 0.8, the sperm density is 10x10 8 Above that, one can use, otherwise discard.
Preparing a cryopreservation liquid, adding PQQ (pyrroloquinoline quinone) with the concentration of 0, 50 mu mol/L, 100 mu mol/L and 150 mu mol/L into M diluent, uniformly mixing with semen, filling into 250 mu L frozen fine tubes, placing into a 4 ℃ refrigerator for balancing for 3 hours, placing into a position 4cm away from a liquid nitrogen surface for liquid nitrogen fumigation, finally placing into the liquid nitrogen for cryopreservation, taking out after three days of freezing, and placing the frozen fine tubes into a 37 ℃ water bath kettle for 30s of thawing and testing, wherein the M diluent is an aqueous solution containing 15% of egg yolk, 6% of fructose and 6% of sodium citrate by mass percent.
3. Quality inspection
3.1 sperm motility
Slide specimens were prepared by pipetting 4. Mu.l of semen, counting under a 400-fold microscope, and repeating three times for each sample with no less than three fields of view. Sperm motility= (number of linear sperm/total number of sperm) ×100%, and the results are shown in table 1.
3.2 plasma membrane integrity and acrosome integrity
Sperm samples were treated using PI, H33342, and PNA mixed staining methods. Firstly, 50 mu L of semen sample is mixed with 10 mu L of PI and 5 mu L H33342 stain, water bath is carried out at 37 ℃ for 15 min, after incubation is finished, PBS is used for washing off the stain, 100 mu L of PBS is used for flushing and suspending sperm sediment, 10 mu L of semen sample is uniformly smeared on a glass slide, the temperature of 37 ℃ is slowly air-dried, 10 mu L of PNA stain working solution is dripped on the air-dried sample, and the mixture is dyed for 30min in a dark place. And after dyeing, placing the glass slide on a constant temperature plate at 37 ℃ for air drying, then dripping 4% paraformaldehyde solution on the sample for fixing for 10 min, and drying. The sample on the slide glass was washed with PBS solution to wash out the staining solution, dried on a thermostatic plate at 37℃and then one drop of glycerol was added dropwise, and after cover glass was covered, it was observed under a fluorescent inverted microscope. 3 visual fields are randomly taken from each sample, the integral state of plasma membranes and acrosomes of about 400 sperms is recorded, the damaged sperms of the plasma membranes are dyed red, the percentage of the integral number of the acrosomes of the sperms to the number of all sperms is evaluated according to the chromogenic state of the sperms, and the calculation integrity is calculated, and the result is shown in table 1.
TABLE 1 Effect of PQQ at different concentrations on the cryopreservation Effect of bovine semen
| PQQ concentration | N | Sperm motility (%) | Plasma membrane integrity (%) | Top body integrity (%) |
| 0µmol/L | 9 | 36.44±0.924 d | 41.82±1.21 c | 46.49±1.16 c |
| 50µmol/L | 9 | 42.276±0.422 c | 46.99±1.79 bc | 56.71±2.19 b |
| 100µmol/L | 9 | 58.404±1.469 a | 62.91±1.47 a | 63.66±1.19 a |
| 150µmol/L | 9 | 48.23±0.905 b | 49.41±3.06 b | 56.57±2.96 b |
Note that: n represents the number of test repetitions. The different letters to the right of the same row of data indicate significant differences (P < 0.05).
4. Antioxidant capacity assay
4.1ROS detection
After collection of cells, probes were loaded: the ROS assay of the semen sample was performed with reference to the instructions of the Solebao reactive oxygen species detection kit. Briefly, add DCFH-DA probe with final concentration of 10. Mu.M, blow and mix gently with a pipette, then incubate the sample in water bath at 38deg.C for 25 min, wash out the impurities in the sample after the reaction with PBS solution, dilute the sperm sample to original volume, add 96-well plate, and detect fluorescence value with enzyme-labeled instrument.
And (3) detection: the sample loaded with the probe after collecting the cells can be detected by a fluorescence spectrophotometer, a fluorescence enzyme-labeled instrument or a flow cytometer, and can also be directly observed by a laser confocal microscope.
Parameter setting: the intensity of fluorescence before and after stimulation was detected in real time or time-point by time using an excitation wavelength of 488nm, an emission wavelength of 525 nm. The fluorescence spectrum of DCF is very similar to FITC, and DCF can be detected with the parameter settings of FITC.
4.2 Sperm MDA, ATP, BCA protein concentration detection
Samples for ATP (adenosine triphosphate), MDA (malondialdehyde) and BCA protein concentration detection were prepared using ATP detection lysate, RIPA (strong) lysate, respectively, to sperm samples washed with PBS for impurities. After ultrasonic crushing in an ice bath environment, the sample is waited at a refrigerator at 4 ℃, and then the upper solution obtained by centrifugation is detected by using an ATP detection kit (Biyun-Tian-Sheng), an MDA (malondialdehyde) detection kit (Biyun-Tian-Sheng) and a BCA protein concentration determination kit (Biyun-Tian-Sheng) respectively.
The protein concentration was determined after sample preparation to facilitate subsequent calculation of MDA and ATP content per protein weight in tissues or cells, and the results are shown in Table 2.
TABLE 2 Effect of PQQ at different concentrations on the cryopreservation Effect of bovine semen
| PQQ concentration | N | ROS (fluorescence value/1 x 10) 6 Sperm cell | MDA（µmol/mg） | ATP（µmol/mg） |
| 0µmol/L | 9 | 221.04±5.15 a | 4.22±0.11 a | 1.26±0.04 b |
| 50µmol/L | 9 | 196.83±3.04 b | 3.68±0.1 b | 1.38±0.02 ab |
| 100µmol/L | 9 | 177.76±5.34 c | 2.74±0.09 d | 1.47±0.04 a |
| 150µmol/L | 9 | 207.58±16.64 ab | 3.05±0.07 c | 1.44±0.08 a |
Note that: n represents the number of test repetitions. The different letters to the right of the same row of data indicate significant differences (P < 0.05).
Compared with a control group after bovine semen is frozen and thawed at ultralow temperature, the sperm motility, plasma membrane integrity, acrosome integrity and ATP content of a PQQ concentration treatment group of 100 mu mol/L are obviously improved (P is less than 0.05); the content of Reactive Oxygen Species (ROS) and Malondialdehyde (MDA) was significantly reduced (P < 0.05). Since the motility of sperm is related to the concentration of ATP in the sperm. Higher levels of Adenosine Triphosphate (ATP) are significantly associated with increases in forward motile sperm rate.
In conclusion, the structure damage and the oxidation damage of the sperms in the freezing process are reduced by treating the cryopreserved sperms with 100 mu mol/L PQQ, so that the activity of the cryopreserved sperms is improved, and the quality of the cryopreserved sperms is greatly improved. PQQ provides a certain technical support for bovine semen preservation.
Example 2
The procedure of this example was the same as in example 1 (but semen was bovine semen at different collection points), except that when preparing the cryopreservation solution, 0 control group, 100 [ mu ] mol/L of PQQ (pyrroloquinoline quinone), 1mmol/L of GSH (glutathione), 2mmol/L of GSH, 3mmol/L of GSH were added to the M diluent, and after mixing with semen, 250 μl of frozen tubule was filled, placed in a 4 ℃ refrigerator for 3 hours, placed at a position 4cm from the liquid nitrogen surface for liquid nitrogen fumigation, finally placed in liquid nitrogen for cryopreservation, taken out after three days of freezing, and thawed in a 37 ℃ water bath pot for 30s to be measured, wherein the M diluent was an aqueous solution containing 15% egg yolk, 6% fructose and 6% sodium citrate by mass, the indexes before cryopreservation were as shown in Table 3, and the indexes after cryopreservation were as shown in Table 4.
TABLE 3 index before cryopreservation
| Project | Control group | 100. Mu. Mol/LPQQ group | 1mmol/L group GSH | 2mmol/L group GSH | 3mmol/L group GSH |
| Sperm motility (%) | 76.00±0.86 d | 86.62±0.55 a | 81.26±0.73 b | 81.93±0.77 b | 78.71±0.15 c |
| Plasma membrane integrity (%) | 86.39±0.72 b | 93.21±0.37 a | 87.74±0.86 b | 86.80±0.68 b | 87.68±0.37 b |
| Top body integrity (%) | 79.28±2.01 c | 91.23±0.42 a | 85.75±1.49 b | 84.67±1.64 b | 84.06±3.34 b |
| ROS (fluorescence value/1 x 10) 6 Sperm cell | 245.24±5.43 a | 173.91±4.44 d | 203.05±5.83 c | 184.65±4.84 b | 198.28±3.71 b |
| MDA（umol/mg） | 3.38±0.12 a | 2.54±0.04 c | 2.88±0.04 b | 2.8958±0.07 b | 2.85±0.08 b |
| ATP（umol/mg） | 7.54±0.05 d | 12.23±0.4 a | 8.80±0.12 c | 10.32±0.46 b | 8.29±0.29 c |
TABLE 4 index after cryopreservation
| Project | Control group | 100. Mu. Mol/LPQQ group | 1mmol/L group GSH | 2mmol/L group GSH | 3mmol/L group GSH |
| Sperm motility (%) | 43.06±0.57 e | 61.10±0.56 a | 55.41±0.64 c | 57.92±0.47 b | 51.56±0.88 d |
| Plasma membrane integrity (%) | 50.27±1.10 d | 69.69±1.44 a | 61.27±0.94 b | 64.22±0.74 b | 59.21±1.26 c |
| Top body integrity (%) | 47.21±2.68 e | 64.41±1.77 a | 56.71±1.42 c | 59.83±0.87 b | 50.98±2.03 d |
| ROS (fluorescence value/1 x 10) 6 Sperm cell | 234.25±3.91 a | 185.73±3.45 d | 223.34±1.91 b | 214.00±1.00 c | 225.83±3.02 ab |
| MDA（umol/mg） | 4.13±0.26 a | 1.96±0.09 c | 3.34±0.21 b | 2.32±0.06 c | 3.46±0.17 b |
| ATP（umol/mg） | 1.10±0.03 c | 1.82±0.18 a | 1.29±0.02 bc | 1.49±0.04 b | 1.23±0.07 bc |
The results show that after the bovine semen is frozen and thawed at ultralow temperature, the sperm motility, plasma membrane integrity, acrosome integrity and ATP content of a PQQ concentration treatment group with the concentration of 100 [ mu ] mol/L are obviously higher than those of a GSH group (P is less than 0.05); the content of Reactive Oxygen Species (ROS) and Malondialdehyde (MDA) was significantly reduced (P < 0.05). The treatment with PQQ showed better results than the treatment with antioxidant GSH.
Claims (1)
1. The application of pyrroloquinoline quinone in reducing the structural damage and oxidative damage of sperms in the freezing process and improving the sperm motility is characterized in that pyrroloquinoline quinone is added before semen freezing,
the freezing process is to add the semen into a freezing preservation solution containing pyrroloquinoline quinone for preservation, wherein the concentration of the pyrroloquinoline quinone in the freezing preservation solution is 100 mu mol/L;
the semen is bovine semen.
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