CN117958253A - Application of limonin, inositol and/or L-proline in pig semen cryopreservation - Google Patents
Application of limonin, inositol and/or L-proline in pig semen cryopreservation Download PDFInfo
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- CN117958253A CN117958253A CN202410142927.2A CN202410142927A CN117958253A CN 117958253 A CN117958253 A CN 117958253A CN 202410142927 A CN202410142927 A CN 202410142927A CN 117958253 A CN117958253 A CN 117958253A
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- semen
- proline
- diluent
- limonin
- inositol
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- 229960002429 proline Drugs 0.000 title claims abstract description 63
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- KBDSLGBFQAGHBE-MSGMIQHVSA-N limonin Chemical compound C=1([C@H]2[C@]3(C)CC[C@H]4[C@@]([C@@]53O[C@@H]5C(=O)O2)(C)C(=O)C[C@@H]2[C@]34COC(=O)C[C@@H]3OC2(C)C)C=COC=1 KBDSLGBFQAGHBE-MSGMIQHVSA-N 0.000 title claims abstract description 61
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 title claims abstract description 60
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses application of limonin, inositol and/or L-proline in cryopreservation of porcine semen. The research of the invention shows that the addition of limonin, inositol and/or L-proline in conventional semen freezing diluent can significantly improve sperm motility, morphological parameters and kinematic parameters after thawing. The three components are added into the semen freezing diluent after being compounded, so that the activity, the membrane integrity, the antioxidant capacity, the in vitro fertilization capacity and the like of the semen after the semen is frozen and thawed can be greatly improved, and the quality of the frozen semen of the pig is remarkably improved.
Description
Technical Field
The invention relates to the technical field of pig semen cryopreservation, in particular to application of limonin, inositol and/or L-proline in pig semen cryopreservation.
Background
At present, the pig semen freezing technology is still in a test stage. Because the sperm motility after the pig semen is unfrozen is poor and the conception rate of the fresh semen can not be reached, the artificial insemination is still mainly carried out on the fresh semen, and the establishment of a sperm gene bank of an excellent pig variety and the convenience of artificial insemination are greatly influenced. Therefore, improving the technology of frozen semen of pigs and improving the quality of frozen semen are one way for promoting the development of pig industry.
The Debao black pig is a good black pig variety with coarse feeding resistance, strong disease resistance and wide adaptability. Mainly distributed in Guangxi Zhuang autonomous region Debao county. Because of the excellent properties of the Debao black pigs, most of the Debao black pigs are used for hybrid improvement work since 90 s of the last century, so that the number of Debao black pigs is drastically reduced, and the number of Debao boars is more endangered. Therefore, research on improving the semen freezing technology of the Debao black pigs and improving the quality of the thawed sperms plays a vital role in the development of the Debao black pigs and the preservation of germplasm resources.
The pig sperm has a special structure, the ratio of cholesterol to phospholipid in a plasma membrane is low, the content of sphingomyelin and phosphatidylethanolamine is high, the outer membrane of the content of cholesterol is larger than that of the inner membrane, the arrangement is irregular, and the characteristics lead to the Debao black pig sperm to be particularly sensitive to cold stress and have high requirements on the operation environment and technical flow. Due to the specificity of the pig sperm membrane, the pig frozen semen is not completely put into production at present.
The frozen diluent is a nutrient and a protective agent for the pig sperm during the freezing and thawing process, and determines the quality of the frozen semen of the pig. The components of the freeze diluent formulation may be divided into nutritional, cryoprotectant and other protectants according to their function. Some components have only a single function, but most components have multiple functions. The conventional freezing diluent mainly comprises glucose, EDTA, potassium chloride, sodium citrate, sodium bicarbonate, penicillin, streptomycin, egg yolk and glycerol. Wherein glucose is mainly used as a nutrient and is used as an energy substance to supplement the energy consumption of sperms, and saccharides also have the functions of impermeable cryoprotectant and maintaining the osmotic pressure of the solution. EDTA is used as chelating agent, and can combine with Ca 2+ and other harmful metal ions in diluted liquid to form stable complex, so as to raise sperm survival rate. Potassium chloride is the osmotic pressure adjustment of the diluent. Sodium bicarbonate and sodium citrate are buffers used to adjust the pH of semen. Penicillin and streptomycin are antibiotics added into the diluent, so that the killing capacity to harmful bacteria can be improved, and the preservation activity of sperms is improved. Egg yolk is a cryoprotectant. Glycerol is a cryoprotectant, and can protect sperm from damage caused by temperature change during freezing and thawing. However, the sperm viability, membrane integrity, antioxidant capacity, in vitro fertilization capacity and the like of the pig semen after thawing the conventional freezing diluent are poor, and the semen is not suitable for the development of the Debao black pigs and the preservation of germplasm resources.
The existing research shows that the oxidation level of the thawed sperm and the integrity of the membrane can be improved by adding an antioxidant and an antifreeze agent into the animal sperm freezing diluent. However, since the absorption and utilization of antioxidants and antifreeze are different from each other in each animal and the way they react with, the improvement effect of antioxidants and antifreeze on frozen semen of each animal is greatly different. Due to its own species specificity, porcine frozen semen requires more stringent antioxidants and antifreeze agents.
The myo-inositol and limonin have strong antioxidation capability. Inositol exists in large quantities in boar seminal plasma and epididymal fluid, and researches show that inositol participates in cell membrane formation and regulating maturation of sperm cells and acrosome reaction. The antioxidant capacity of limonin is 12.5-17.2 times of that of vitamin C, so that the in vitro maturation quality of the oocyte and bovine oocyte of a mouse is obviously improved, and the oxidative stress capacity is improved. L-proline is a naturally occurring amino acid with osmoprotectant and antioxidant capacity, has small molecular weight, is very soluble in water, has neutral pH and no toxicity, is often used as a cryoprotectant, and is widely applied to the fields of biological, medical and agricultural research. The addition of low-level L-proline in cryopreservation of sheep sperm, donkey sperm, human mesenchymal stem cells and human endothelial cells obviously improves the survival rate of cells after freezing and thawing, and the L-proline can play a protective role in normal-temperature preservation of the porcine semen through the antioxidant capacity of the L-proline, so that the application of the L-proline in the cryopreservation of the porcine semen to improve the sperm viability, membrane integrity, antioxidant capacity and in-vitro fertilization capacity of the thawed porcine semen is not seen at present.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings in the prior art and provide the application of limonin, inositol and/or L-proline in the cryopreservation of porcine semen.
A second object of the present invention is to provide the use of limonin, inositol and/or L-proline as additives in the preparation of a frozen dilution of porcine semen.
A third object of the present invention is to provide a porcine semen freezing diluent.
The fourth object of the invention is to provide a preparation method of the pig semen freezing diluent.
A fifth object of the present invention is to provide the use of said porcine semen freezing diluent.
The above object of the present invention is achieved by the following technical solutions:
In order to improve the activity, membrane integrity, oxidation resistance, in vitro fertilization capacity and the like of the sperm after the frozen semen of the pig is thawed, the quality of the frozen semen of the pig is improved. According to the invention, limonin, inositol and L-proline are respectively added into the conventional basic semen freezing diluent to detect the vitality, morphology and kinematic parameters of the thawed sperms, and the result shows that the appropriate addition of limonin, inositol and L-proline can obviously improve the vitality, morphological parameters and kinematic parameters of the thawed sperms. Further, the three components are compounded, and oxidation indexes such as membrane integrity, acrosome integrity, mitochondrial membrane potential and the like of the thawed sperm are detected by adding limonin, inositol and L-proline into the conventional basic sperm freezing diluent, so that the quality, fertilization capacity and the like of the thawed sperm are measured, and the result shows that the added new diluent greatly improves the activity, membrane integrity, antioxidation capacity, in-vitro fertilization capacity and the like of the sperm after the frozen and thawed pig semen, and improves the quality of the frozen pig semen.
Thus, the present invention provides the following new uses for limonin, inositol and L-proline:
The application of limonin, inositol and/or L-proline in the cryopreservation of the porcine semen can improve the sperm motility, membrane integrity, antioxidant capacity and in-vitro fertilization capacity of the unfrozen porcine semen and improve the quality of the porcine frozen semen by using limonin, inositol and/or L-proline in the dilution process of the cryopreservation of the porcine semen.
The application of limonin, inositol and/or L-proline as additives in the preparation of the pig semen freezing diluent can improve the sperm viability, membrane integrity, oxidation resistance and in vitro fertilization capacity of the thawed pig semen and improve the quality of the pig frozen semen by using the pig semen freezing diluent added with limonin, inositol and/or L-proline.
The application of limonin, inositol and L-proline in improving sperm motility, membrane integrity, antioxidant capacity and in vitro fertilization capacity of thawed pig semen is provided.
Further, the concentration of limonin is 100-150 mu mol/mL.
Preferably, the limonin is added at a concentration of 150 μmol/mL.
Further, the inositol is added at a concentration of 0.03 to 0.12mmol/mL.
Preferably, the inositol is added at a concentration of 0.06 to 0.12mmol/mL.
Further preferably, the inositol is added at a concentration of 0.09 to 0.12mmol/mL.
More preferably, the inositol is added at a concentration of 0.09mmol/mL.
Further, the L-proline is added at a concentration of 50 to 150mmol/mL.
Preferably, the L-proline is added at a concentration of 100mmol/mL.
The invention also provides a pig semen freezing diluent which contains 100-150 mu mol/mL limonin, 0.03-0.12 mmol/mL inositol and 50-150 mmol/mLL-proline.
The pig semen freezing diluent comprises exogenous antioxidant limonin, endogenous antioxidant inositol and small molecule cryoprotectant L-proline. Compared with the prior art, after the exogenous antioxidant limonin, endogenous antioxidant inositol and small molecule cryoprotectant L-proline are added into the conventional semen freezing diluent, the limonin is used as the exogenous antioxidant and the inositol is matched as the endogenous antioxidant, so that the oxidative stress capability of frozen sperms can be improved; the L-proline is added to reduce the use of antifreeze glycerol, and the new diluent greatly improves the activity, membrane integrity, oxidation resistance, in vitro fertilization capacity and the like of sperms after the semen of pigs is frozen and thawed. The frozen diluent for the porcine semen effectively improves the quality of the semen after thawing, obviously reduces the oxidative stress reaction of the semen, and improves the integrity of the semen after thawing and the in-vitro fertilization capacity. The semen freezing diluent can be used for freezing and preserving the pig semen, especially the semen of a Debao black pig, and is beneficial to protecting germplasm resources.
Preferably, the pig semen freezing diluent contains 100-150 mu mol/mL limonin, 0.06-0.12 mmol/mL inositol and 50-150 mmol/mL L-proline.
Further preferably, the pig semen cryodiluent contains 150. Mu. Mol/mL limonin, 0.09mmol/mL inositol and 100 mmol/mLL-proline.
The invention also provides a preparation method of the pig semen freezing diluent, which is to add limonin, inositol and L-proline into the prepared semen freezing diluent.
Further, the pig semen freezing diluent contains glucose, EDTA, potassium chloride, sodium citrate, sodium bicarbonate, penicillin, streptomycin, egg yolk and glycerol.
Further, the preparation method of the pig semen freezing diluent comprises the step of adding limonin, inositol and L-proline into the prepared semen freezing diluent.
Further, the pig semen freezing diluent is a basic semen freezing diluent I solution and a basic semen freezing diluent II solution which contain 150 mu mol/mL of limonin, 0.09mmol/mL of inositol and 100mmol/mL of proline, and the basic semen freezing diluent II solution is prepared by adding glycerol to the basic semen freezing diluent I solution which contains 150 mu mol/mL of limonin, 0.09mmol/mL of inositol and 100mmol/mL of proline, and the glycerol concentration is 6%. For example: to 94mL of base semen freeze diluent I was added 6mL of glycerol.
As a preferred embodiment, the base semen freezing diluent I liquid is: glucose 2.96g, ethylenediamine tetraacetic acid 0.1g, KCl 0.06g, sodium citrate 0.48g, naHCO 3 0.1.1 g, penicillin sodium 0.048g, streptomycin sulfate 0.08g, dissolved in 80mL ultra-pure water, pH adjusted to 7.2, and then added with 20mL sterile egg yolk to a volume of 100mL, and stored at 4 ℃.
The basic semen freezing diluent II solution is as follows: 6mL of glycerol was added to 94mL of the base semen freeze diluent I and stored at 4 ℃.
The freezing preservation of the porcine semen mainly relates to the steps of semen collection, quality identification, dilution and balancing, filling, freezing, preservation, thawing, artificial insemination after dilution and the like. The pig semen freezing diluent is used for dilution and balancing, and by adopting the pig semen freezing diluent for dilution and balancing in the link, compared with the conventional pig freezing diluent, the pig semen freezing diluent can greatly improve the vitality, membrane integrity, oxidation resistance, in-vitro fertilization capacity and the like of the sperm after freezing and thawing the pig semen, and improve the quality of the pig frozen semen.
Therefore, the invention also provides the application of any of the pig semen freezing diluent in improving the quality of pig frozen semen, and the pig semen freezing diluent is used for collecting pig semen and quality identification, and then sequentially diluting and balancing, filling and freezing and preserving; the dilution is performed by adopting the pig semen freezing diluent of any one of the above.
Further, the pig is a Debao black pig.
Compared with the prior art, the invention has the following beneficial effects:
The research of the invention shows that the addition of limonin, inositol and/or L-proline in the conventional semen freezing diluent can significantly improve the sperm motility, morphological parameters and kinematic parameters after thawing. The three components are added into the semen freezing diluent after being compounded, so that the activity, the membrane integrity, the antioxidant capacity, the in vitro fertilization capacity and the like of the semen after the semen is frozen and thawed can be greatly improved, and the quality of the frozen semen of the pig is remarkably improved.
Drawings
FIG. 1 shows the effect of semen freezing dilutions with different amounts of limonin on post-thawing sperm motility, morphological parameters and kinetic parameters.
Figure 2 shows the effect of semen freezing dilutions with different amounts of inositol on sperm motility, morphological parameters and kinematic parameters after thawing.
FIG. 3 shows the effect of semen freezing dilutions with different levels of L-proline on sperm motility, morphological parameters and kinetic parameters after thawing.
FIG. 4 is a graph showing the detection of post-frozen sperm motility, morphology and kinetic parameters using a sperm freezing dilution with limonin, L-proline and inositol added.
FIG. 5 shows the results of the detection of mitochondrial membrane potential of frozen sperm treated with a semen freezing dilution supplemented with limonin, L-proline and inositol.
FIG. 6 shows the results of the detection of antioxidant capacity of frozen sperm cells treated with a semen freezing dilution with limonin, L-proline and inositol.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
1. Experimental animal
The Debao black pig sperm adopted by the invention are all from the Guangxi Zhuang autonomous region livestock and poultry variety improvement station.
2. Solution preparation
Basic semen freezing diluent I: 2.96g glucose, 0.1g ethylenediamine tetraacetic acid, 0.06gKCl g sodium citrate, 0.48g NaHCO 3, 0.048g penicillin sodium and 0.08g streptomycin sulfate are weighed and dissolved in 80mL ultrapure water, the pH is adjusted to 7.2, and then 20mL sterile egg yolk is added to a volume of 100mL and stored at 4 ℃.
Basic semen freezing diluent II: 9mL of glycerol was added to 91mL of the base semen freeze diluent I and stored at 4 ℃.
Preparing a thawing solution: 2.6g glucose, 0.8g sodium citrate, 0.12g NaHCO 3, 0.24g ethylenediamine tetraacetic acid, 0.9g Hepes, 0.25g BSA, 0.06g penicillin sodium, 0.1g streptomycin sulfate were weighed out in 100mL ultra pure water, and the pH was adjusted to 6.8,4 ℃for preservation.
Preparing a hypotonic solution: 1.351g of fructose and 0.735g of sodium citrate dihydrate were weighed out and dissolved in 100mL of ultrapure water and stored at 4 ℃.
Oocyte wash (CCM): 9.5g TCM199, 2.2g NaHCO 3, 1.2g Hepes, 3% FBS, 0.06g penicillin, 0.1g streptomycin were weighed out in 1L ultra pure water and stored at 4 ℃.
Oocyte maturation fluid (PM): 0.550g glucose, 0.069g cysteine, 0.100g sodium pyruvate, 1X 10 4. Mu.g EGF, 16IU Pregnant Mare Serum Gonadotropin (PMSG), 8IU Human Chorionic Gonadotropin (HCG), 10% FBS+10% follicular fluid were weighed out in 50mL TCM199 and stored at 4 ℃.
Embryo culture fluid (PZM-3): 0.6312g NaCl、0.0746g KCl、0.2106g NaHCO3、0.0048g KH2PO4、0.0048g MgSO4、0.0617g calcium lactate, 0.0022g sodium pyruvate, 0.0292g glutamine, 0.0546g hypotaurine, 2mL amino acid solution, 1mL optional amino acid, 0.0066g penicillin, 0.005g streptomycin, 0.3g BSA were weighed out in 100mL ultrapure water and stored at 4 ℃.
0.1% Hyaluronidase (HYT): 100mg HYT was weighed into 100mL CCM and stored at 4 ℃.
Semen wash (mTBM): 0.66g NaCl, 0.02236g KCl, 0.11347g CaCl 2·2H2 O, 0.11346g Tris Base, 0.242g glucose, 0.05502g sodium pyruvate, 0.11g BSA were weighed out in 100mL ultra pure water and stored at 4 ℃.
Semen (PF): 0.0388g caffeine was dissolved in 100mL of semen and stored at 4 ℃.
Tissue culture medium (TCM 199), fetal Bovine Serum (FBS) were purchased from Gibico, USA and the remaining reagents were purchased from Sigma-Aldrich, unless otherwise specified.
3. Semen freezing specific operation
1. Semen collection and preliminary assessment
Collecting semen by hand-held semen collection method, collecting Carnis Sus Domestica essence with excellent reproductive performance, delivering the collected semen to semen freezing chamber within 30min, and transferring into graduated sterile enzyme-free centrifuge tube; and observe the semen state, the normal semen is milky white or milky yellow, slightly fishy; and then, a proper amount of sperm is measured and dripped on a glass slide, and the glass slide is subjected to microscopic examination, so that the sperm is frozen after the living rate of the sperm before the freezing is observed to be higher than 80 percent.
2. Dilution and equilibration:
(1) Counting the number of the sperm before freezing, then putting the sperm before freezing into a constant temperature box at 17 ℃ for balancing for 30min, then centrifuging for 15min at 17 ℃ at 2500rpm, and discarding the supernatant.
(2) The volumes required for frozen dilutions I and II (i+sperm pellet volume: ii=1:1) were calculated from the number of sperm obtained so that sperm density became 1×10 9/mL.
(3) Slowly pumping the frozen diluent I solution into the sediment along the wall of the centrifuge tube, so that the sediment is completely dissolved in the semen frozen diluent I solution. And (3) rapidly placing the semen added with the semen freezing diluent I into a 5-DEG C constant temperature cabinet for cooling (3.5 h). And after the semen is cooled to 5 ℃, adding the frozen diluent II solution, and placing the mixture in a 4-DEG C incubator for continuous balancing for 30min.
(4) Filling and freezing: sucking the balanced semen into 0.25mL frozen semen tube, sealing with sealing powder, freezing with program freezer, cooling at-2 deg.C/min at 4-1 deg.C, at-30 deg.C/min at-1 to-25 deg.C, at-30 deg.C/min at-25 to-140 deg.C, and storing in liquid nitrogen.
4. Thawing and subsequent detection of semen
0.25ML of frozen seminiferous tubules were clamped from liquid nitrogen with forceps, placed in a 37 ℃ water bath for resuscitation for 30s, and detected after adding thawing solution.
1. Detection of post-freezing sperm motility, morphology and kinematic parameters
Pig sperm motility, morphology and kinetic parameters were measured using a sperm analyzer (French IVM company, model CEROSII). 10 mu L of thawed sperm are dripped on a preheated glass slide, 5 fields of view are read from each sample, and the samples are repeatedly detected for 10 times and then averaged.
2. Detection of sperm plasma membrane integrity after freezing
Taking 100 mu L of unfrozen semen, putting the semen into 1mL of hypotonic solution (incubating for 5min at 37 ℃), incubating for 30min in a water bath kettle at 37 ℃ after uniformly mixing, taking 15 mu L of the incubated semen, dripping the 15 mu L of the incubated semen on a cell counting plate, observing the bending condition of the tail part of the sperms under a 400X optical microscope, experimentally calculating 200 sperms, and repeating for 5 times. Sperm with intact plasma membrane has its tail bent due to swelling in hypotonic solution; sperm plasma membrane integrity = bent tail sperm/total number of counted sperm x 100%.
3. Detection of post-freeze sperm acrosome integrity
The observation was stained with PNA-FITC fluorescent dye (purchased from Sigma-Aldrich). The specific method comprises the steps of taking 50 mu L of thawed sperms, washing the sperms with PBS for 3 times, adding PNA-FITC fluorescent dye working solution, fully mixing the sperms, incubating the sperms in a water bath at 37 ℃ for 30min in a dark place, rapidly photographing and observing the sperms under a fluorescence microscope, repeating each group for three times, and counting more than 200 sperms each time; sperm acrosome integrity = number of sperm that did not fluoresce green/total number of sperm counted x 100%.
4. Detection of post-frozen sperm nuclear DNA integrity
The dye was used for observation by 0.01% of acridine orange dye (A0 dye, available from Soy Corp.). The specific method comprises the steps of taking 50 mu L of thawed sperms, washing the sperms with PBS for 3 times, adding diluted acridine orange staining solution, fully mixing the mixture, incubating the mixture in a water bath at 37 ℃ for 10min in a dark place, rapidly photographing and observing the mixture under a fluorescence microscope, repeating each group for 3 times, and counting more than 200 sperms each time; sperm nuclear DNA integrity = number of green fluorescent sperm/total number of counted sperm x 100%.
5. Detection of mitochondrial membrane potential of frozen sperm
The sample was stained with JC-1 staining solution (purchased from Biyun Tian Co.). The specific method comprises the steps of preparing JC-1 working solution and 1X dyeing buffer solution according to the specification; washing 50 mu L of thawed sperms with PBS for 3 times, adding the prepared JC-1 working solution, fully and uniformly mixing, incubating for 20min in a water bath at 37 ℃ in a dark place, and adding a pre-cooled 1X staining buffer solution for 2 times; then 600g, centrifuging for 3min, discarding supernatant, re-suspending with a proper amount of staining buffer solution, rapidly photographing and observing under a fluorescence microscope, repeating each group for 3 times, wherein each sperm count is more than 200; mitochondrial membrane potential = red fluorescence intensity/green fluorescence intensity.
6. Detection of antioxidant capacity of frozen sperm
Specific methods of using a Catalase (CAT) assay kit, a total antioxidant capacity (T-AOC) assay kit, a micro Malondialdehyde (MDA) assay kit, a glutathione peroxidase (GSH-PX) assay kit, and a superoxide dismutase (SOD) assay kit (all of which are purchased from Nanjing built biosystems) are exemplified by the specification.
7. In vitro fertilization
Pig ovaries were taken from the yu Ban Lu slaughterhouse in nanning, stored in a saline incubator sterilized at about 30 ℃ and sent to the laboratory rapidly. After cleaning, extracting medium-sized follicles by using a 10mL disposable syringe, and selecting a cumulus oocyte complex which is wrapped by a cumulus and has complete and uniform cytoplasm to mature for 44 hours in vitro in oocyte maturation liquid; the mature oocytes are digested with hyaluronidase to remove cumulus cells surrounding the oocytes. The digested oocytes were washed 3 times in wash semen, 15 oocytes were transferred into 50. Mu.L of fertilized droplets, the thawed semen was placed in receiver semen in an incubator at 38.5℃upstream for 1h, followed by centrifugation at 1500rpm for 5min, the supernatant was discarded, and sperm concentration was adjusted to 1X 10 6 per mL with fertilized liquid. Then placing the sperms and the oocytes in a culture box with 5% CO 2, 38.5 ℃ and saturated humidity for culturing for 6 hours, taking out fertilized eggs, gently blowing off residual sperms on the surfaces of the fertilized eggs in PZM-3 solution, placing 15 fertilized eggs in 30 mu L of PZM-3 liquid drops for continuous culturing, and counting the cleavage rate and blastula rate during the culturing period.
EXAMPLE 1 concentration screening of limonin, inositol and L-proline additions in semen frozen dilutions
1. The method is characterized by comprising the following steps:
(1) Basic semen freeze diluent I solution containing 0. Mu. Mol/mL, 50. Mu. Mol/mL, 100. Mu. Mol/mL, 150. Mu. Mol/mL, 200. Mu. Mol/mL limonin. Specifically, limonin with corresponding concentration is added into the prepared basic semen freezing diluent I solution of every 100 mL.
(2) Basic semen freezing diluent I solution containing 0. Mu. Mol/mL, 0.03. Mu. Mol/mL, 0.06. Mu. Mol/mL, 0.09. Mu. Mol/mL, 0.12. Mu. Mol/mL inositol. Specifically, inositol with corresponding concentration is added into the prepared frozen diluent I liquid of every 100mL of basic semen.
(3) Basic semen freezing dilution I solution containing 0. Mu. Mol/mL, 50. Mu. Mol/mL, 100. Mu. Mol/mL, 150. Mu. Mol/mL L-proline. Specifically, L-proline with a corresponding concentration is added to the prepared frozen diluent I liquid of every 100mL of basic semen.
2. According to the method, semen freezing and thawing are carried out, and the motility, morphology and kinematics parameters of the frozen semen are detected, so that the influence of the basic semen freezing diluent I solution added with different contents of limonin, inositol or L-proline on the motility, morphology parameters and kinematics parameters of the thawed semen is evaluated, and therefore proper addition concentration is screened.
The effect results of the basic semen freezing diluent I solution added with different limonin contents on the sperm motility, morphological parameters and kinematic parameters after thawing are shown in figure 1, and the vitality, morphological parameters and kinematic parameters of the rest groups are higher than those of the control group except that 50 mu mol/mL limonin is added into the semen freezing diluent. As shown in fig. 1-a, at the limonin addition concentration of 150 μmol/mL, the activity was significantly higher than the control group and other concentration groups (P < 0.05); as shown in fig. 1-B, at the limonin addition concentration of 150 μmol/mL, the bent tail was significantly lower than the control group and other concentration groups (P < 0.05); distal and proximal mass drops were significantly lower than control, 50 μmol/mL and 200 μmol/mL (P < 0.05), with no significant difference from the 100 μmol/mL group (P > 0.05); the tail of the coil is significantly lower than that of the group with 100 mu mol/mL (P < 0.05), and no significant difference is generated from other addition groups (P > 0.05); as shown in fig. 1-C, at the limonin addition concentration of 150 μmol/mL, VCL, ALH, DAP kinematic parameters were not significantly different from the control group (P > 0.05), DSL, WOB, VAP, LIN, VSL, STR kinematic parameters were significantly higher than the control group (P < 0.05); in conclusion, the limonin is added into the frozen diluent, so that the activity, morphology and kinematic parameters of the frozen sperm can be obviously improved, the optimal addition concentration is 150 mu mol/mL, and the limonin 150 mu mol/mL is determined as the optimal addition amount for subsequent experiments.
The effect results of the basic semen freezing diluent I solution added with different inositol content on the sperm motility, morphological parameters and kinematic parameters after thawing are shown in figure 2, showing that the motility is remarkably improved (P < 0.05) after inositol is added into the semen freezing diluent; the difference was most pronounced at an added concentration of 0.09mmol/mL (FIG. 2-A); FIG. 2-B shows that the 0.06mmol/mL group has less tail-bending significance than the control group and the 0.03mmol/mL group (P < 0.05), and that there is no significant difference from the 0.09mmol/mL, 0.12mmol/mL group (P > 0.05). The tail of the coil of the group with 0.09mmol/mL and 0.12mmol/mL is obviously lower than that of the group with 0.06mmol/mL (P < 0.05); the distal mass drop in the 0.09mmol/mL, 0.12mmol/mL group was significantly lower than that in the control group, 0.03mmol/mL group (P < 0.05), and there was no significant difference from the 0.06mmol/mL group (P > 0.05); proximal mass drops were significantly lower in the 0.06mmol/mL group than in the control and other groups (P < 0.05). Fig. 2-C shows that the VCL, ALH, BCF parameter of the 0.09mmol/mL group showed an increasing trend in terms of the kinematic parameter compared to the control group, but no significant difference (P > 0.05), DAP, DSL, WOB, VAP, LIN, VSL, STR was significantly elevated (P > 0.05) compared to the control group. In summary, 0.09. Mu. Mol/mL inositol was determined as the optimal addition for subsequent testing.
The influence results of the basic semen freezing diluent I solution added with different contents of L-proline on the sperm motility, morphological parameters and kinematic parameters after thawing are shown in figure 3, the motility is remarkably improved (P < 0.05) after the L-proline is added into the semen freezing diluent, and the difference is most remarkable when the adding concentration is 100mmol/mL (figure 3-A); 100mmol/mL group bent tail significantly lower than control and other groups (P < 0.05), distal mass drop significantly lower than control and 150mmol/mL (P < 0.05), no significant difference from 50mmol/mL group (P > 0.05), tail significantly lower than 50mmol/mL group (P < 0.05), no significant difference from control and 150mmol/mL group (P > 0.05) (FIG. 3-B); the VCL, ALH, DAP, DSL, WOB, LIN, VSL, STR of the 100mmol/mL group showed a significant increase in kinematic parameters (P < 0.05) compared to the control group, BCF, VAP showed an increasing trend, but no significant difference (P > 0.05) (fig. 3-C). In summary, 100mmol/mL of L-proline was determined as the optimal addition for subsequent experiments.
EXAMPLE 2 addition of limonin, L-proline and inositol to semen frozen diluent
1. Experiment set control group, MYO group, L-proline group, lim group and MYO+L-proline+lim group
Control group: basic semen freezing diluent I liquid and basic semen freezing diluent II liquid which do not contain limonin, inositol and L-proline;
MYO group: basic semen frozen diluent I solution containing 0.09mmol/mL inositol. Specifically, inositol with corresponding concentration is added into the prepared frozen diluent I liquid of every 100mL of basic semen. Basic semen freezing diluent II: to 91mL of the basic semen freeze diluent I solution containing 0.09mmol/mL inositol was added 9mL of glycerol.
Group of L-prolines: a basic semen frozen dilution I solution containing 100mmol/mL L-proline. Specifically, L-proline with a corresponding concentration is added to the prepared frozen diluent I liquid of every 100mL of basic semen. Basic semen freezing diluent II: to 91mL of the basic semen freeze diluent I containing 100mmol/mL L-proline was added 9mL of glycerol.
Lim group: a basic semen freezing diluent I solution containing 150 mu mol/mL limonin. Specifically, limonin with corresponding concentration is added into the prepared basic semen freezing diluent I solution of every 100 mL. Basic semen freezing diluent II: to 91mL of base semen freeze diluent I containing 150. Mu. Mol/mL limonin was added 9mL of glycerol.
Lim group and myo+l-proline+lim group: a basic semen freezing diluent I solution containing 150 mu mol/mL limonin, 0.09mmol/mL inositol and 100mmol/mL L-proline is specifically prepared by adding limonin, inositol and L-proline with corresponding concentrations into every 100mL of the prepared basic semen freezing diluent I solution. Basic semen freezing diluent II: to 94mL of basal semen freeze diluent I solution containing 150. Mu. Mol/mL limonin, 0.09mmol/mL inositol and 100mmol/mL L-proline was added 6mL glycerol.
2. Freezing semen, thawing, and detecting
(1) Detection of post-freezing sperm motility, morphology and kinematic parameters as shown in fig. 4, after combined use of the three, the post-freezing sperm motility was significantly higher than that of the control group and the addition group alone (P < 0.05) (fig. 4-a); the bent tail and the rolled tail have no significant difference (P < 0.05) from the control group and the independent addition group, the far-end mass drop is lower than that of the control group and the MYO addition group (P < 0.05), and the far-end mass drop has no significant difference (P > 0.05) from the L-proline addition group and the Lim addition group; proximal mass drops were significantly lower than control and Lim-added groups, with no significant difference (P > 0.05) from MYO-added and L-proline added groups (fig. 4-B); in terms of kinematic parameters, the three compatible use group ALH, DAP, VAP, DSL, WOB, LIN, VSL, STR had a significant improvement (P < 0.05) with the single addition group, and VCL, BCF did not have significant differences (P > 0.05) (fig. 4-C). The combination of the three components can improve the activity of the sperm after freezing, improve morphological parameters and kinematic parameters, and is superior to the single addition group.
(2) The results of the detection of the integrity of the plasma membrane of the sperm after freezing are shown in table 1, and the plasma membrane integrity rate, the acrosome membrane integrity rate and the nuclear DNA integrity rate of the three combined use groups are all obviously superior to those of a control group and a single addition group (P < 0.05), namely 34.40%, 76.01% and 64.46%, respectively. The three are combined to obviously improve the plasma membrane integrity, the acrosome membrane integrity and the nuclear DNA integrity rate of the sperm after thawing.
TABLE 1 sperm plasma membrane integrity, acrosome membrane integrity, nuclear DNA integrity after freezing
(3) As shown in FIG. 5, the mitochondrial membrane potential of the frozen sperm is remarkably improved (P < 0.05) compared with the control group and the L-proline group after the three groups are combined, and has no remarkable difference (P > 0.05) from the MYO addition group and the Lim addition group, but the mitochondrial membrane potential is higher than that of the single addition group. The combination of the three can improve the mitochondrial membrane potential and the mitochondrial function of sperms.
(6) The detection result of the antioxidant capacity of the frozen sperms is shown in figure 6, and after the three are combined, CAT content shows an ascending trend (P < 0.05) compared with a control group and a single addition group (figure 6-A); GSH-PX had increased significance (P < 0.05) compared to the control and L-proline added groups, no significant difference (P > 0.05) from the MYO added and Liim added groups (fig. 6-B); T-AOC and SOD levels were significantly elevated compared to the control group (P < 0.05), but not significantly different from the addition group alone (P > 0.05) (fig. 6-C and 6-E); MDA content tended to decrease significantly (P < 0.05) compared to the control group, with no significant difference (P > 0.05) from the addition group alone (FIG. 6-D). The combined use of the three components improves the oxidative stress capability of the thawed sperms, and the effect is better than that of a single addition group.
(7) The results of in vitro fertilization are shown in Table 2, and the cleavage rate of IVF embryos of the control group is 54.81% and the blastocyst rate of the control group is 2.25%; the Lim group and MYO+L-proline+lim group (experimental group) have the cleavage rate of 68.81% and the blastocyst rate of 7.31%, and the cleavage rate and the blastocyst rate of the experimental group are both obviously improved (P < 0.05), which indicates that the semen freezing diluent added with limonin, inositol and L-proline can obviously improve the in vitro fertilization capacity of sperms.
TABLE 2IVF embryo development
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The application of limonin, inositol and/or L-proline in pig semen cryopreservation.
2. The application of limonin, inositol and/or L-proline as additives in preparing frozen dilution of pig semen.
3. The use according to claim 2, wherein the limonin is added at a concentration of 100 to 150 μmol/mL.
4. The use according to claim 2, characterized in that the inositol addition concentration is 0.03-0.12 mmol/mL.
5. The use according to claim 2, wherein the L-proline is added at a concentration of 50 to 150mmol/mL.
6. A frozen diluent of pig semen is characterized by comprising limonin 100-150 mu mol/mL, inositol 0.03-0.12 mmol/mL and L-proline 50-150 mmol/mL.
7. The frozen swine semen diluent of claim 6, comprising 150 μmol/mL limonin, 0.09mmol/mL inositol, and 100mmol/mL L-proline.
8. The frozen swine semen diluent of claim 6, comprising glucose, EDTA, potassium chloride, sodium citrate, sodium bicarbonate, penicillin, streptomycin, egg yolk, glycerol.
9. A method of preparing a porcine semen freeze diluent according to claim 6 or 7 wherein limonin, inositol and L-proline are added to the prepared semen freeze diluent.
10. Use of the porcine semen freezing diluent of claim 6 or 7 to enhance the quality of porcine frozen semen.
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