NL2034272A - Long-acting diluent for boar semen preservation at normal temperature based on oregano essential oil - Google Patents
Long-acting diluent for boar semen preservation at normal temperature based on oregano essential oil Download PDFInfo
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- NL2034272A NL2034272A NL2034272A NL2034272A NL2034272A NL 2034272 A NL2034272 A NL 2034272A NL 2034272 A NL2034272 A NL 2034272A NL 2034272 A NL2034272 A NL 2034272A NL 2034272 A NL2034272 A NL 2034272A
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- semen
- essential oil
- normal temperature
- sperm
- boar semen
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- 238000004321 preservation Methods 0.000 title claims abstract description 83
- 239000003085 diluting agent Substances 0.000 title claims abstract description 53
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- 239000000463 material Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 3
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- DZYNKLUGCOSVKS-UHFFFAOYSA-N epigallocatechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3cc(O)c(O)c(O)c3 DZYNKLUGCOSVKS-UHFFFAOYSA-N 0.000 description 2
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- 210000002966 serum Anatomy 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 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 description 2
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- 230000000844 anti-bacterial effect Effects 0.000 description 1
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- RECUKUPTGUEGMW-UHFFFAOYSA-N carvacrol Chemical compound CC(C)C1=CC=C(C)C(O)=C1 RECUKUPTGUEGMW-UHFFFAOYSA-N 0.000 description 1
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- 235000007746 carvacrol Nutrition 0.000 description 1
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- 150000002215 flavonoids Chemical class 0.000 description 1
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- WYXXLXHHWYNKJF-UHFFFAOYSA-N isocarvacrol Natural products CC(C)C1=CC=C(O)C(C)=C1 WYXXLXHHWYNKJF-UHFFFAOYSA-N 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
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Classifications
-
- 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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- 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 present invention discloses a long-acting diluent for boar semen preservation at normal temperature and a method of improving the quality of boar semen stored at normal temperature. 5 The long-acting diluent for boar semen preservation at normal temperature includes oregano essential oil. The method of improving the preservation quality of boar semen at normal temperature is mixing the oregano essential oil with the diluted boar semen, and then storing it at normal temperature; the concentration of the oregano essential oil in the mixed solution is 0.06- 0.08 mg/mL. The present invention systematically explores the morphological and structural 10 changes and antioxidant properties of sperm after adding oregano essential oil during the preservation process of boar semen at normal temperature, and reveals that oregano essential oil can improve the quality of semen and prolong the preservation time of semen. The sperm motility rate reached 70.50% on the 9th day when the semen was stored at normal temperature. The present invention is the first time that oregano essential oil is used as a boar semen additive, 15 and the research results provide a new theoretical basis for the development and use of a diluent for boar semen preservation at normal temperature.
Description
LONG-ACTING DILUENT FOR BOAR SEMEN PRESERVATION AT NORMAL
TEMPERATURE BASED ON OREGANO ESSENTIAL OIL
S [0001] The present invention belongs to the technical field of pig breeding and reproduction, 1m particular to a long-acting diluent for boar semen preservation at normal temperature based on oregano essential oil.
[0002] Compared with traditional insemination technology, artificial insemination technology can increase the use efficiency of boars by about 10 times, while reducing the number of boars and effectively reducing breeding costs. Through the combination of artificial insemination technology and modern breeding technology, high-quality breeding boars are selected, and the semen of excellent breeding boars are sent to various production plants to establish genetic links between farms, realize rapid gene promotion, and accelerate the rate of genetic progress.
However, there are often many factors which would affect the quality of boar semen preservation in production, such as the breed, age and individual difference among boars, etc. The accumulation of reactive oxygen species generated by sperm metabolic activities and bacterial contamination of semen are also key factors which would reduce the quality, for the reason that excessive reactive oxygen species impair the integrity of the plasma membrane of sperm and acrosome structure, leading to decreasing in sperm motility rate and also restricting long-distance transportation of semen. How to maintain high-quality boar semen has become an important link in the development of artificial insemination technology, establishing high-quality semen coverage in multiple factories, overcoming the space limitations of artificial insemination technology, and enabling excellent boars to give full play to their advantages. Therefore, adding appropriate antioxidants and bacteriostatic agents is the key factor for the preservation of boar semen at normal temperature.
[0003] Origanum plants of the Lamiaceae family are widely known for their herbal properties and aromatic medicinal properties, and play a very important role in the economy. Worldwidely,
origanum is used in traditional medicine in countries such as Turkey, Greece and ltaly, mainly used to solve respiratory disorders, rheumatoid arthritis, nutritional disorders and urinary problems. Oregano essential oil (OHO) is a plant essential oil extracted from origanum, which is mainly composed of aromatic phenols, flavonoids and terpenoids, and its chemical components
S are rich in thymol and carvacrol. In the past two decades, OEQ has gradually become a research hotspot in anti-infection, playing the role of antioxidant and antibacterial respectively.
[0004] In animal husbandry, OEO 1s often used as a bacteriostatic agent and antioxidant, and 18 widely used in feed additives for livestock and poultry. It has been reported that when oregano essential oil is used as a feed additive, the balance of the intestinal microflora could be significantly adjusted, thereby improving the intestinal digestion and absorption function of laying hens and improving the feed utilization rate.
[0005] However, there is currently no application of oregano essential oil in the preservation of boar semen at normal temperature.
[0006] The object of the present invention is to provide a new use of oregano essential oil.
[0007] The new use of the oregano essential oil provided by the present invention is its use in the preservation of boar semen at normal temperature or the use in the preparation of long-acting diluent for boar semen preservation at normal temperature.
[0008] Another object of the present invention is to provide a long-acting diluent for boar semen preservation at normal temperature.
[0009] The long-acting diluent for boar semen preservation at normal temperature provided by the present invention includes the diluent powder for a boar semen preservative, and further includes oregano essential oil.
[0010] The diluent powder for the boar semen preservative can be the American VIM boar semen diluent powder, the German MZ diluent powder for the boar semen preservative, the
Xinzuan diluent powder for the boar semen preservative, the Danish KRUUSE diluent powder for the boar semen preservative, etc.
[0011] Another object of the present invention is to provide a method of improving the preservation quality of boar semen at normal temperature.
[0012] The method of improving the preservation quality of boar semen at normal temperature provided by the present invention includes the following steps: mixing an oregano essential oil
S with diluted boar semen and stored at normal temperature.
[0013] In the present invention, the diluted boar semen is obtained by diluting the freshly collected boar semen with a diluent powder solution for the boar semen preservative commonly used in the art, wherein the density of the boar semen is diluted to 450-550 million/mL.
[0014] The diluent powder for the boar semen preservative can be the American VIM boar semen diluent powder, the German MZ diluent powder for the boar semen preservative, the
Xinzuan diluent powder for the boar semen preservative, the Danish KRUUSE diluent powder for the boar semen preservative, and the like.
[0015] The sperm motility rate of the diluted boar semen is greater than 80%.
[0016] In the present invention, the normal temperature usually ranges from 15°C to 17°C, specifically 17°C.
[0017] In the present invention, in the mixture of the oregano essential oil and the diluted boar semen, the concentration of the oregano essential oil is 0.02-0.10 mg/ml, preferably 0.06-0.08 mg/ml.
[0018] In the present invention, the oregano essential oil is added in the form of an oregano essential oil aqueous solution, and the concentration of the oregano essential oil aqueous solution can be 0.02-0.1 mg/ml.
[0019] The present invention systematically explores the morphological and structural changes and antioxidant properties of sperm after adding oregano essential oil during the preservation process of boar semen at normal temperature, and reveals that oregano essential oil can improve the quality of semen and prolong the preservation time of semen. The sperm motility rate reached 70.50% on the 9th day when the semen was stored at normal temperature. oregano essential oil in the concentration range that has a protective effect on sperm has no obvious inhibitory effect on bacteria. An oregano essential oil is used as a boar semen additive for the first time in the present invention and the research results provide a new theoretical basis for the development and use of diluent for boar semen preservation at normal temperature.
[0020] The present invention has the following beneficial technical effects: 1. The present invention analyzes the change trend of semen pH value, the sperm motility rate, the integrity of the plasma membrane of sperm and the integrity of the acrosome of sperm after adding OEO to the semen diluent of boar semen stored at normal temperature for 1,3,5,7, 9, and 10 days. The addition of OEO can improve sperm motility, sperm structure, and improve the effect of semen preservation, and the addition of 0.06 mg/mL has the best effect. 2. Adding 0.06-0.08 mg/mL OEO to the semen diluent can effectively increase the SOD enzyme activity and CAT activity in semen, and reduce the level of ROS in semen.
FIG 1 shows the integrity of the plasma membrane of boar sperm; the tail of the plasma membrane of boar sperm is curled after being treated with Host solution, the yellow arrow marks the sperm with intact plasma membrane, and the white arrow marks the sperm with damaged plasma membrane. Scale bars=210 pm.
Fiz. 2 shows the morphology of boar sperm after hypotonic treatment.
FIG. 3 shows the staining of the acrosome of boar sperm; the acrosomes of boar sperm were stained with FITC-PNA and showed bright and complete green fluorescence, the yellow arrows mark sperm with intact acrosomes, and white arrows mark sperm with acrosome defects; sperm nuclei show blue fluorescence after staining with DAPL Scale bars=80 um.
FIG. 4 shows the morphology of the acrosome after staining of boar sperm.
FIG 5 shows the effect of oregano essential oil on ROS content in boar semen, Note: *%* means significant difference (p<0.05}, **** means extremely significant difference {(p<0.01}).
FIG. 6 shows the effect of OEG on CAT content in boar semen. Note: * means significant difference (p<0.05), ** means significant difference (p<0.05}, *** means extremely significant difference (p<0.01};
FIG 7 shows the effect of OEO on SOD content in boar semen. Note: * means significant 5 difference (p<0.0S), ** means significant difference {p<0.05).
[0021] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from open commercial sources unless otherwise specified. [00221 Example 1, the effect of oregano essential oil on the preservation effect of boar semen at normal temperature
I Experimental materials and methods 1.1 Experimental material 1.1.1 Experimental semen [00231 The boar semen used in the experiment came from Beijing Shunxin Agricultural Duroc
Original Breeding Pig Farm. The mid-section semen of 3 healthy 10-month-old Duroc boars was collected by the hand-grip method. The semen collection personnel visually detected the color of fresh semen and tested the quality of the semen. After the semen was considered to be normal,
KRUUSE commercial diluent powder {at No 950031} was used to prepare commercial diluted semen, placed in an incubator for 2 hours, and then sent to the laboratory, and stored in a 17°C incubator. The semen with a sperm motility rate greater than 80% and above can be used for follow-up experiments.
[0024] Preparation of commercial diluent: 2-3 hours before using diluent of KRUUSE pig semen diluent powder (product model: Cat. No 950031, short-acting diluent), 48g of powder was taken and dissolved in 1000 ml of sterile distilled water, shaken slightly until completely dissolved; the diluent was placed in a constant temperature water bath and heated to about 35°C before use.
[0025] Preparation of commercial diluted semen: obtained by diluting the above normal boar semen with a commercial diluent, wherein the density of boar sperm was diluted to 450-550 million/mL. 1.1.2 Experimental reagents
S [0026] oregano essential oi, standard product, purity 298% (Shanghai Yuanye Company); 6.4- 8.4 range of precision pH test paper, polylysine-treated adhesive glass slide, cover glass; sodium citrate, fructose (Sinopharm Chemical Reagent Co, Ltd); FITC-PNA, IMG (Sigma), DAPI 10mL {Solarbio) 1.2 Experimental method 1.2.1 Experimental Design
[0027] After the commercial diluted semen was transported to the laboratory, 5 experimental groups and 2 control groups were set up. Among them, 0.02 mg/mL, 0.04 mg/mL, 0.06 mg/mL, 0.08 mg/mL, 0.10 mg/mL of an oregano essential oil solution were respectively added to the commercial diluted semen of the experimental group, and five replicates were set for each 14 concentration; The same volume of distilled water was added to the commercial diluted semen in the control group, the blank group was the commercial diluted semen stock. Stored ina 17°C incubator and mixed slowly every 12h. On the Ist, 3rd, 5th, 7th, 9th and 10th days of boar semen preservation at normal temperature, the semen pH, sperm motility, the integrity rate of the plasma membrane of sperm and the integrity rate of the acrosome of sperm were detected to explore the effect of oregano essential oil on boar semen preservation at normal temperature. 1.2.2 Preparation of the solution (1) Preparation of oregano essential oil solution: the stock solution was prepared with distilled water to a concentration of 8.1 mg/ml. and stored at -20°C. (2) Preparation of hypotonic solution: 0.49 g of sodium citrate and 0.9 g of fructose were accurately weighed and dissolved in 100 mL of ultrapure water. The osmotic pressure of the solution was 150 mOsm. (3) FITC-PNA working solution: the stock solution was diluted by 1:50 with PBS, then aliquoted, and stored at -20°C for later use.
(4) DAPI working solution: the stock solution was diluted by 1:10 with PBS, then aliquoted, and stored at -20°C for later use. 1.2.3 Semen pH value detection
[0028] Determination was carried out using precision pH test paper in the range 6.4-8 4. 10 ul. of semen was sucked with 4 pipette and dripped onto the test paper, and then compared with the colorimetric bar of the test paper. 5 random drop repetitions were performed in each group. 1.1.2.4 Sperm motility rate
[0029] Detections of the sperm motility rate were performed on days 1, 3,5, 7, 9, and 10 of semen preservation using computer-assisted sperm analysis (CASA). The operation steps were strictly in accordance with the experiment operation steps provided by CASA. (1) 100 ul of semen was sucked with a pipette into a sterilized 1.5mL centrifuge tube, water bathed at 37°C for 10min, and the thermostatic stage was turned on to preheat at 37°C. (2} After the water bath, 10 ul. of semen was sucked by a pipette, dripped onto a glass slide, covered with a cover glass and placed under a microscope. (3) The CASA instrument was turned on, the microscope was adjusted, and five fields of view were randomly selected for detection. The number of sperm in each field of view was about 200. Each group of samples was randomly selected 5 times for measurement, and the data was finally recorded. 1.2.5 Integrity rate of the plasma membrane of sperm
[0030] The integrity rate of the plasma membrane of boar sperm on the Ist, 3rd, 5th, 7th, 9th and 10th days of semen stored at 17°C was determined by a Hypo-osmotic Swelling Test (HOST) test. Simultaneously, sperm counts were performed using Image] software. After the sperm was incubated in the hypotonic solution for 30min, the tail changed. The tail of sperm with intact plasma membrane was curved due to swelling. The formula for calculating the integrity of the plasma membrane of sperm can be based on the rate of sperm with curved tail = the number of sperm with curved tail / the total number of sperm counted * 100%.
(1} 200 ul of hypotonic solution was sucked with a pipette and preheated in a constant temperature water bath at 37°C for Smin, 20 pL of semen was pipetted into 200 ul of hypotonic solution, and placed in a constant temperature water bath at 37°C for 30min. (2) 10 pL of semen was sucked and placed under a 400x optical microscope, and three clear fields of view were randomly selected for photography. (3) ImagJ software was used to perform sperm counts and to calculate the ratio of the number of sperms with curved tails to the number of total sperms. 1.2.6 Integrity rate of the acrosome of sperm
[0031] Fluorescein isothiocyanate-peanut agglutinin (FITC-PNA ) fluorescent staining method was used to detect the acrosomes of boar sperm stored at 17°C for the 1st, 3rd, 5th, 7th, 9th and 10th days. At the same time, the boar sperm was counterstained with DAPI fluorescent dye to exclude false positives caused by impurities and non-cellular substances. Sperm counts were performed using Image! software. (1) 20ul of semen was dripped onto a glass slide, spread evenly with a pipette tip, and air- dried naturally. (2} An appropriate amount of anhydrous methanol was sucked by a pipette and dripped onto the air-dried sample glass slide, so that the anhydrous methanol completely covered the sample, and placed in the air to dry naturally. (3) 20 ul of FITC-PNA working solution was pipetted and evenly dripped onto the sample glass slide to cover the sample, and then the glass slide was placed in a humid box, and incubated in a 37°C incubator for 40 nun in the dark. (4) The incubated sample glass slides were taken out and washed with PBS repeatedly for S- 10min each time to remove the floating color on the surface of the glass slide. (5) 20 ul of DAPI working solution was dripped to stain sperm nuclei to cover the sample, and placed in a hunudified box for staining at room temperature for 10 nun. (6) After the staining, a small amount of glycerol was dripped to the staining area, covered with a cover glass, stored in a humidified box, and observed with a fluorescence microscope. Three or more clear fields of view were randomly selected for photography, and the whole process was protected from light. After FITC-PNA staining, the acrosome front of sperm with intact acrosome showed complete green bright fluorescence, and the acrosome front of sperm with damaged acrosome showed incomplete or no fluorescence. Sperm nuclei showed blue fluorescence after staining with DAPL (7} Imag] software was used for sperm counting. 1.3 Statistical analysis
[0032] The data of this experiment were statistically processed by Microsoft Excel, and SPSS 26 software was used for one-way (ANOVA) analysis of variance. Duncan's multiple comparisons were used to test the significance of differences. The final data results were expressed in the form of mean + standard deviation, and p < 0.05 meant significant difference, p < 0.01 meant extremely significant difference. Graphs were made using GraphPad Prism 7. 2 Experimental results 2.1 The effect of oregano essential oil on the pH value of boar semen stored at normal temperature
[0033] The pH value of boar semen stored at 17°C on the 1st, 3rd, Sth, 7th, 9th and 10th days was detected. The results were shown in Table 1. The treatment group added with oregano essential oil was added with 0.02 mg/mL, 0.04 mg/mL, 0.06 mg/mL, 0.08 mg/mL, 0.10 mg/ml, the control group was added with the same amount of distilled water, and the blank group was commercial diluted semen stock without the addition of distilled water and oregano essential oil.
The pH value of each group was made basic statistics. The results showed that the overall pH value of boar semen during normal temperature preservation was in the range of 7.0 to 8.0, and the sperm living environment remained basically stable. The semen was stored for 1-5 days, and the pH values of the treatment group, the control group and the blank group were all maintained at 7.0-7.5. The pH value of the semen without oregano essential oil began to increase from the 5th day of preservation to 7.5. The pH of the semen in the control group changed from the 3rd day, ranging from 7.2 to 7.5. On the 7th to 10th day of semen preservation, the pH value of boar semen after adding the oregano essential oil increased from 7.2 to 7.5, and the pH value of the control and blank groups increased to 8.0 on the 9th day.
i0
Table 1 Effects of different concentrations of oregano essential oil on pH of boar semen stored at normal temperature
QED Semen pH f 7.2 72 7.3 7.5 7.5 TSR.
Conral 7.2 7.2~7.5 72-75 7.5 7.5 7.58.0 0.02 10-72 70-72 7.2 7.3 75 7.58.0 0.04 TOT 70-732 7.2 F218 TITS 7.5 9,06 10-72 T72 1.2 TTS FINS 75 {1.08 7.2 7.2 7.2 LISTS 1.5 7.5
IG 1.2 7.2 7.2 7.2~7.5 TA 7538.0 2.2 The effect of oregano essential oil on the sperm motility rate of boar semen stored at normal temperature
[0034] The effect of adding the oregano essential oil on the sperm motility rate of boar semen stored at 17°C on the st, 3rd, 5th, 7th, 9th and 10th days was shown in Table 2 below. The
CASA instrument detection showed that the sperm motility rate of boars decreased with the increase of preservation time, and the sperm motility rate remained above 80% from 1 to 7 days. it showed a significant downward trend from the 9th day, and invalid sperm appeared on the 10th day. After adding the oregano essential oil to the boar semen diluent, the sperm motility rate of boars was improved to varying degrees. (On the 1st to 9th days of semen preservation, the sperm motility rate of the treatment group supplemented with 0.06 mg/mL was significantly higher than that of the control group and blank group without oregano essential oil (p<<0.05). On the Ist to 3rd day of preservation, the sperm motility rate of the supplemented 0.06 mg/mL group was significantly higher than that of the control and blank groups (p<0.05), followed by the 0.08 mg/mL and 0.04 mg/mL groups; on the 5th day of preservation, the sperm motility rate of the 0.06 mg/mL group was 93.65%, which was still the highest among the groups; on the 7th day of preservation, there was no significant difference between the groups; on the 9th day of il preservation, the sperm motility rate was the lowest when the concentration of the oregano essential oil was 0.02 mg/mL, and the sperm motility rate when the concentration was 0.1, 0.08 and 0.04 mg/ml. was also significantly different from the control and blank groups { p<0.05), the sperm motility rate of the 0.06 mg/mL group was still the highest. On the 10th day of
S preservation, when the concentration of the oregano essential oil in the semen reached 0.1 mg/mL, the sperm motility rate was significantly higher than that in the other treatment groups and the control group (p<0.05), but the sperm motility rates in each group were all below 50%.
Table 2 The effect of different concentrations of oregano essential oil on the sperm motility rate of boar semen stored at normal temperature
OBO Sperm Motility Tate (9%) (mgimly 1d 3d Sd 7d ad Wid ù QI042092° GO DIE TE OOSSKI0IP SESSLT AN 59562069 AS20560
Control GLTRE)2S 9L7302% SBI SS BAIR:0.13 S9.15:128¢ IS GOLD 0.02 SLE2.680 QLO4a2.6% SORTHIBOY RRASELTI ALE6053Y 12,05. 9D 0.04 SI OTL038® G4.0040.87F 2.134092 SSAELLAD 62.08:0460 17,95 07° 0.06 7494020 GOITER 2365063 ST 2la2 455 TOS0:12S 2.264047 0.08 GI65H088 GLA OON DOGOELIID SS 1413 GRSTEOSIE 224441390 2.3 The effect of oregano essential oil on the integrity of the plasma membrane of sperm of boar semen preserved at normal temperature [00351 The effect of adding the oregano essential oil on the integrity of the plasma membrane 14 of sperm on the Ist, 3rd, Sth, 7th, 9th, and 10th days of boar semen preservation at 17°C was shown in Table 3 below. After being treated with hypotonic solution, the sperm tails showed different shapes {as shown in FIG. 2) under the microscope. The curled state of the tail was the sperm with complete plasma membrane (A in FIG, 2), while the sperm tail with incomplete plasma membrane were not curved (B in FIG. 2}. It can be seen from Table 3 that the integrity of the plasma membrane of sperm decreased with the increase of preservation time when boar sperm was stored at 17°C for 1-10 days, and the integrity of the plasma membrane decreased the fastest on the 10th day of preservation. The integrity rate of the plasma membrane of sperm after the addition of the oregano essential oil remained above 70% for 1-5 days. Among them, the treatment group with an oregano essential oil concentration of 0.06 mg/mL had the highest
S integrity rate of the plasma membrane of sperm from 1 to 9 days of semen preservation compared with all groups. On the first day of semen preservation, the integrity rate of the plasma membrane of sperm of the treatment group with a concentration of 0.06 mg/ml. was the highest at 84.64%, which was significantly different from that of the control group (p<0.05). With the increase of the concentration of the oregano essential oil, the integrity rate of the plasma membrane of sperm did not increase. On the 10th day, only the 0.06 mg/mL and 0.10 mg/mL groups in the treatment group added with oregano essential oil achieved 50% integrity of the plasma membrane, which were 50.5% and 50.57%, respectively. Compared with the other groups, the integrity rate of the plasma membrane of the original sperm in blank group was the highest at 51 39%, but there was no significant difference with the 0.06 mg/mL treatment group.
Table 3 Effects of different concentrations of oregano essential oil on the integrity of the plasma membrane of boar semen stored at normal temperature
OBO Integrity of the plasma membrane (2%) imginl) Id id Sd Td 4d {0d & READY TEIJIN SDN IP 6385:088 SDI TN SL WD KE
Conte ERTEGEN TIOMDAN TOLEN BITE STIRS 47134085 {2 BITS 0 TRAI2:0.23Y 203500410 BST ODIS 4065030
IM S2I7ADATN TEIGE TES0028Y SRSLY GLE AN 498119 $3.06 R4.64:041% BI 74 TEST IDG GTI SQA dS 2372042 SOSDIEN TEIGE DI TSI GL 206] JI ZOE RTE 018 |. ELTA A DSP Se satan Janene
Note: =$. The results were expressed as "mean + standard deviation”; different superscripts (a, b, ¢} in the same column indicated significant differences (p<0.05).
i3 2.4 The effect of oregano essential oil on the integrity of the acrosome of sperm of boar semen preserved at normal temperature
[0036] The effect of adding the oregano essential oil on the sperm motility rate of boar semen stored at 17°C on the 1st, 3rd, 5th, 7th, 9th and 10th days was shown in Table 4 below. After
S staining with FITC-PNA and DAPL pictures were taken and counted. In FIG. 4, B was the normal acrosome of sperm with FITC-PNA staining, showing bright and complete green fluorescence, and A was the non-fluorescent or fluorescent-deficient sperm with incomplete acrosome. It can be seen from Table 4 that the integrity of the acrosome of sperm decreases with the increase of preservation time, from about 90% to about 57%, a drop of about 30% when the boar sperm was stored at 17°C for 1-10 days. The integrity rate of acrosome was above 80% from 1 to 5 days, and decreased by about 10% on the 7th day. The addition of the oregano essential oil improved the integrity rate of the acrosome of sperm. The experiment showed that the integrity rate of the acrosome of sperm was the highest when the concentration of oregano essential oil was 0.06 mg/ml, followed by the 0.04 mg/mL group, which was significantly different from the control group {(p<0.05). Compared with the control group, the integrity rate of the acrosome of sperm in the 0.06 mg/ml group was about 3% higher than that in the control group on the Ist to 5th day of semen preservation, On the 7th day of preservation, the integrity rate of the acrosome of sperm in the 0.06 mg/mL group was about 7% higher than that of the control group, on the 9th day of preservation, the integrity rate of the acrosome was about 10% higher than that of the control group, and on the 10th day of preservation, the integrity rate of the acrosome of sperm with a concentration of 0.10 rag/mL was the lowest, and with the increase of the concentration of the oregano essential oil, the integrity rate of acrosome showed a downward trend.
Table 2-4 Effects of different concentrations of oregano essential ofl on the integrity of the acrosome of boar sperm
OEQ Inteerity of the acrosome {40} {mnl} id 3d Sd 7d Gd 10d { SOAVE RRIS IDSN RT RTE TA TT TIGO ST GI JI2ORRS 376104
Control SPEEN RRSSSDANS RT 374043 SOOTY 67 B421.389 57.8660" 0.02 $9 laid BR20a044% SR0330.77 TORSMIT T243409% 5R.03a0.64% 0.04 1,540 R0.33:3046% SOTRA0TS ER05a01810 75263078) 80.3430.4660 4.480 GI 97S23 BL32a036% VOTI STB TTA 6542 0E G3O4.30e ND 7420} SSITIO RRD 7E DAD GIS 67340487 ST AGIT
UI BOO 28 NN IBN eld SATZ ATN TARTIO KES 65642037 54534050
Note: n=5, The results were expressed as "mean + standard deviation”; different superscripts (a, b, c) in the same column indicated significant differences (p<0.05}.
[0037] The results of this experiment showed that there was no significant change in the pH value of boar semen stored at 17°C, indicating that the sperm was 10 a stable living environment.
In this experiment, an oregano essential oil was added as an antioxidant to boar semen diluent.
After adding the long-acting diluent, the sperm metabolic activity was in a state of inhibition during the storage period of 1-7 days, and the number of dead sperm was also less, so the sperm motility rate was above 80%. After a long time of energy consumption, the number of effective sperms began to decrease significantly from the 9th day. At this time, the added oregano essential oil played its role. Compared with the control group and the blank group, the 6.06 mg/mL oregano essential oil group had a significant increase in the role of the integrity of the plasma membrane and the integrity of the acrosome. Although the use of oregano essential oil in boar semen had not been reported yet, compared with Zhang Lingjtao's research on epigallocatechin {Zhang Lingjiao et al. Effects of epigallocatechin gallate on the preservation of boar semen at normal temperaturelJ] Journal of Livestock Ecology, 2022, 43{02}. 41-7 1, the preservation effect of oreganc essential oil on semen was much better than that of epigallocatechin for 1-5 days of semen preservation. In Zhao (31's research on gallate [Zhao Ot.
Study on the effect of gallate on the preservation of bear semen at normal temperature [D1];
Northwest A& F University, 2019], although the sperm motility rate of gallate on the 10th day of semen preservation was higher than that of the oregano essential oil, the integrity of the plasma membrane of sperm and the integrity of the acrosome were not as effective as oregano essential oil. 5 3 Summary
[0038] In this experiment, Duroc boar semen was used, and after adding the oregano essential oil, the semen was stored in a 17°C incubator. The pH value of semen, the integrity of the plasma membrane of sperm, and the integrity of the acrosome of sperm were analyzed at 1,3, 5,7, 9, and 10 days, and the following conclusions were obtained: (1) the pH range of semen was 7.0 to 8.0 when stored at normal temperature for 1 to 10 days. (2) the addition of 0.06 mg/mL oregano essential oil significantly improved the sperm motility rate. The sperms motility rate reached 70.50% on the 9th day of semen preservation. (3) the addition of 0.06 mg/mL oregano essential oil significantly smproved the integrity of the plasma membrane of sperm and the integrity of acrosome.
[0039] Example 2, the effect of oregano essential oil on the antioxidant properties of boar semen stored at normal temperature
I Experimental materials and methods 1.1 Experimental material 1.1.1 Experimental semen: the same as that in 1.1.1 of Example 1 1.1.2 Experimental reagents: ROS content detection kit, SOD activity detection kit, and
CAT activity detection kit were purchased from Nanjing Hancheng, and they were strictly operated according to the operation method of Nanjing Jiancheng kits. 1.2 Experimental method 1.2.1 Experimental Design
[0040] After the commercial diluted semen was transported to the laboratory, 5 experimental groups and 2 control groups were set up. Among them, 0.02 mg/mL, 0.04 mg/mL, 0.06 mg/mL, 0.08 mg/mL, 0.10 mg/mL of an oregano essential oil solution were added to the commercial diluted semen of the experimental group. The same volume of water was added to the
S commercial diluted semen in the control group; the blank group was the commercial diluted semen stock. The semen was stored in a 17°C incubator and mixed slowly every 12h. On the
Ist, 3rd, Sth, 7th, Sth and 10th days of boar semen preservation at normal temperature, the ROS content, SOD and CAT activities in semen were measured to explore the effect on the antioxidant properties of boar semen. 1.2.2 Detection of ROS content in boar sperm cells
[0041] ROS Reactive Oxygen Species Assay Kit (Cat. No: E004-1-1 Chemifluorescence 100T-500T}, and DCFH-DA probe were used to detect the level of reactive oxygen species in boar semen. DCFH-DA working solution was diluted at a ratio of 1:1000. (1} The semen was taken out from the incubator at 17°C, and 100 pL. of semen was sucked with a pipette and added to a centrifuge tube. After centrifugation at 1000g for 5 minutes, and the supernatant was removed. {2y After 200 pL of PBS was added, it was gently pipetted 2-3 times, centrifuged at 1000g for 5 min, and the supernatant was removed. (3) 500 ul DCFH-BDA working solution was added and resuspended, placed in a 37°C water bath for 35 minutes, and a baffle was used to protect from light. During the incubation, the solution was inverted and mixed once every 3-5 minutes so that the
DCFH-DA probe and sperm cells were in full contact. (4) After the water bath, the cells were centrifuged again at 1000 g for 5 min, and washed twice with PBS to remove the DCFH-DA solution that did not enter the sperm cells. (5) After washing, PBS was added to resuspend, and the samples were added to 96-well plate and detected using a fluorescence microplate reader. The excitation wavelength was set to 488 nm and the emission wavelength was set to 525 nm. 1.2.3 CAT enzyme activity detection
[0042] Boar sperm catalase content was detected using the CAT detection Kit (Cat. No. A007- 1-1 Ammonium Molybdate Method). The operation steps were shown in the following table. The test tubes were numbered in advance, 0. 1mL of sample and IniL of reagent 1 were added, and then all test tubes were put into a 37°C water bath and preheated for 3-5min. During the
S detection, 0.1 mL of reagent 2 was added to the test tube, and immediately mixed. At the same time, it was timed accurately and placed in a water bath at 37°C. When the reaction reached 60 seconds, reagent 3 was added immediately (termination of the reaction), and mixed. The second and third tubes were tested in turn, and finally reagent 4 was added to all the test tubes and mixed. A multifunctional microplate reader was used for detection, wavelength: 405 nm.
Calculation formula: Serum (plasma) CAT activity (U/mL) = AAX271/V sample/TxN (T: reaction time, 60 seconds; N: diluent factor of the sample before testing).
Co] Control tube | Assay tube
Reagent 1 (preheat at 37°C) 1.0 | 1.0 {mL)
Reagent 2 (prebeat at 37°C) | 0. ve
Immediately mixed, timing, and accurately reacted at 37°C for 1 minute (60 seconds) 0! of
Mixed well, wavelength 405nm, optical diameter 0.5¢m, double distilled water to zero, measured the absorbance value of each tube, calculated AA=A control A measurement 1.2.4 SOD enzyme activity detection
[0043] The detection was carried out according to the instructions of the SOD assay kit (Cat.
No. A001-3 WAT-1 method). The operation table was as follows. A multifunctional microplate reader was used, and the wavelength was set to: 450nm. Calculation formula: 1. SOD inhibition rate {24)={A control-A control blank)-( A test-A test blank) {A control-A control blank)x100%. 2.
SOD activity (U/mL) = SOD inhibition rate/50% x diluent ratio of the reaction system (0.24mL/0.02mL} * diluent ratio of the sample before testing.
Control wells Control blank | Assay wells | Assay blank wells | wells
Serum to be | - 20 | 20 tested (ul)
Distilled water | 20 20 (ul)
Enzyme working | 20 20 | - solution {pL}
Enzyme Diluent | - 20 - | 20 (WL)
Substrate | 200 200 200 | 200 application solution (mL) 1.3 Data Analysis
[0044] The data of this experiment were processed after Microsoft Excel statistics, and then
SPSS 26 software was used for one-way (ANOVA) analysis of variance, Duncan's multiple comparisons were used to test the significance of differences, and the final data were expressed in the form of mean + standard deviation, and p<0.05 meant significant difference, p<0.01 meant extremely significant difference. Graphs were analyzed using GraphPad Prism7. 2 Experimental results
2.1 The effect of oregano essential oil on the ROS content of boar semen stored at normal temperature
[0045] After the boar semen was stored at normal temperature and added with different concentrations of oregano essential oil, the results of the detection of ROS content in sperm cells
S onthe 1st, 3rd, Sth, 7th, and 9th days were shown in FIG. 5. Fluorescence microplate reader detection showed that when the oregano essential oil was not added to the semen, the ROS content in sperm cells increased with the increase of storage time. On the first day of semen preservation, the treatment group with an oregano essential oil concentration of 0.06 mg/mL had the least ROS content in sperm cells, which was significantly different from the control group {p<0.05}), and there was an extremely significant difference with the blank group without oregano essential oil{p<0.01). On the 3rd day of preservation, there was an extremely significant difference between each treatment group and the control group (p<0.01), and there was also an extremely significant difference with the blank group (p<0.01). Among them, the treatment group with a concentration of 0.08 mg/mL had the lowest ROS level. On the 5th day of preservation, the ROS level in sperm cells of the 0.08 mg/mL treatment group was still the lowest, followed by the 0.06 mg/mL treatment group. On the 7th day of preservation, the level of
ROS in sperm cells showed an obvious upward trend, but compared with the treatment group and the blank group, the ROS level in the sperm cells of the treatment group with a concentration of 0.08 mg/mL remained low, and there was a very significant difference ( p<0.01}. After 9 days of storage, the ROS content in each treatment group showed an obvious upward trend, but there was almost no difference between the groups, and there was still an extremely significant difference compared with the control group and the blank group (p<0.01). 2.2 The effect of oregano essential oil on CAT activity of boar semen preserved at normal temperature
[0046] FIG. 6 showed the detection of CAT content in semen stored at 17°C on the Ist, 3rd, 5th, 7th, and 9th days by adding different concentrations of OEO to the boar semen diluent.
Graph analysis was performed using Graphpadp prism software. The CAT content in the semen without the addition of oregano essential oil decreased significantly with the increase of preservation time. It can be seen from the figure that when the semen was stored for 3-5 days, the decreasing trend was the most obvious. The CAT content in the semen supplemented with 0.06 mg/mL OEO was the highest during the 1-9 days of semen preservation. On the first day of preservation, there was no significant difference between the treatment group, the control group
S and the blank group, but by comparing the mean values, it could be seen that the 0.06 mg/mL group had the highest CAT content in the semen, followed by the 0.04 mg/mL group. On the 3rd day of preservation, the CAT content in each group was not significantly different from that on the 1st day. Compared with the control group, the CAT content in the semen of the 0.06 mg/mL group had no significant difference but was significantly higher than that of the blank group {p<0.05), and secondly, the 0.08 mg/mL treatment group also had a significant difference compared with the blank group (p<0.05). On the 5th day of preservation, there was a significant difference between the treatment groups and the control group (p<0.05}). It can be seen that the antioxidant effect of GEO on semen was more significant at this time. On the 7th day of preservation, the content of CAT in the semen of each treatment group began to show a significant downward trend, but it was still significantly higher than that of the control group (p<0.05). On the 9th day of preservation, the content of CAT in semen decreased significantly, and there was no difference between the groups, but the preservation effect of the treatment group added with an OEO concentration of 0.06 mg/mL was still better. 2.3 The effect of oregano essential oil on the SOD activity of boar semen preserved at normal temperature
[0047] FIG. 7 showed the detection of SOD content in semen stored at 17°C on the 1st, 3rd, 5th, 7th, and 9th days by adding different concentrations of OEQ to the boar semen diluent. After statistical analysis, it can be seen that the SOD content in the semen of the treatment groups with concentrations of 0.06 mg/mL and 0.08 mg/mL was higher. At the same time, the two treatment groups were compared with the blank group and the control group by Graphpadp prism software.
The SOD activity in semen of oregano essential oil decreased with the increase of preservation time. A in Fig. 7 showed the SOD content compared with the blank group, and B in Fig 7 showed the SOD content compared with the control group. It can be seen that the treatment group with 0.08 mg/mL semen preserved for 1-5 days showed significant difference with the blank group and the control group (p<t0.05), and had the highest SOD content in the semen of each group. Among them, on the 3rd day of preservation, the SOD content in each treatment group increased compared with that on the Ist day. On the Sth day of preservation, the SOD
S content in semen began to decrease, and the SOD content in the 0.06 mg/mL and 0.08 mg/mL treatment groups was significantly higher than that in the blank group and the control group (p<0.05). On the 7th day of preservation, there was no significant difference between the treatment group and the blank group and the control group. By comparing the average values, it was found that the SOD content in the 0.08 mg/mL treatment group was still the highest among all groups. On the Sth day of preservation, the SOD content decreased significantly among the groups, but almost the same.
[0048] During the storage of semen at normal temperature, sperm would be affected by various factors and cause stress. For example, the temperature change during transportation, the temperature difference during transportation should be minimized in the experiment. At the same time, in order to prevent sperm agglutination, when the sperm was mixed upside down, it may stress the sperm and exacerbated the increase of ROS level. While protecting DNA integrity of sperm and improving fertilization capacity, antioxidants also protected sperm cell membranes and acrosomes from damage. The content of ROS in semen was positively correlated with the preservation days of semen, but the activity of antioxidant enzymes decreased with the decrease of preservation days. The important antioxidant enzymes in the SOD and CAT antioxidant systems in semen were SOD enzymes that mainly protect the quality of semen. This experiment showed that on the 3rd day when oregano essential oil was added to the semen, the level of ROS in sperm cells with concentrations of 0.06 mg/mL and 0.08 mg/mL decreased compared with that on the 1st day, and the content of CAT had little change compared with that on the 1st day, and the SOD content in the semen increased compared with that on the 1st day. This may be due to the influence of transportation, experiment operation, ambient temperature and other factors on the 1st day of semen preservation and a large amount of ROS was generated, and at this time, the sperm initiated the antioxidant mechanism to produce antioxidant enzymes to protect itself from lipid peroxidation. oregano essential oil started to play an antioxidant role from the 3rd day, and increased the activities of antioxidant enzymes SOD and CAT in semen to reduce the level of
ROS. On the 7th day, all the indicators showed a significant downward trend, the reason of which might be that the antioxidant capacity of OEQ failed. It was found that when the concentration of OEQ increased to 0.08 mg/mL, the contents of CAT and SOD increased, the
S content of ROS decreased, and the preservation effect was better, but the level of ROS did not decrease significantly when the concentration increased to 0.10 mg/mL, which may also be related to the OEO addition dose effect. Therefore, the experiment in this example showed that the addition of OEO to the diluent of boar semen had an improved effect on semen preservation, and can significantly increase the activities of CAT and SOD, and reduce the level of ROS.
Comprehensive comparison, the optimal addition concentration of OEO was 0.06 mg/mL, followed by 0.08 mg/mL. 3 Summary
[0049] Adding OEO to boar semen diluent improved the quality of semen at normal temperature, and when the concentration was 0.06-0.08 mg/mL, the content of ROS in semen was the lowest, and the activities of CAT and SOD were the highest.
[0050] The present invention systematically explored the morphological and structural changes and antioxidant properties of sperm after adding oregano essential oil during the preservation process of boar semen at normal temperature, and revealed that oregano essential oil can improve the quality of semen and prolong the storage time of semen. The sperm motility rate reached 70.50% on the 9th day when the semen was stored at normal temperature. The oregano essential oil in the concentration range that has protective effect on sperm had no obvious inhibitory effect on bacteria. The present invention was the first ime that oregano essential oil was used as a boar semen additive, and the research results provided a new theoretical basis for the development and use of a diluent for boar semen preservation at normal temperature.
[0051] In the following paragraphs, clauses are provided:
Clause 1. Use of an oregano essential oil in the preservation of boar semen at normal temperature.
Clause 2. Use of an oregano essential oil in the preparation of a long-acting diluent for boar semen preservation at normal temperature.
Clause 3. A long-acting diluent for boar semen preservation at normal temperature, comprising diluent powder for a boar semen preservative, characterized in that the long-acting diluent for
S boar semen preservation at normal temperature further comprises an oregano essential oil.
Clause 4. A method for improving the preservation quality of boar semen at normal temperature, comprising the following steps: mixing an oregano essential oil with dituted boar semen and storing at normal temperature.
Clause 5. The method according to clause 4, characterized in that the diluted boar semen is obtained by diluting freshly collected boar semen with a diluent powder solution of a boar semen preservative, wherein a density of the boar semen is diluted to 450 million/mL 550 million/mL.
Clause 6. The method according to clause 4 or 5, characterized in that a sperm motility rate of the diluted boar semen is greater than 80%.
Clause 7. The method according to any one of clauses 4-6, characterized in that the normal temperature is usually 15-17°C.
Clause &. The method according to any one of clauses 4-7, characterized in that in a mixture of the oregano essential oil and the diluted boar semen, a concentration of the oregano essential oil is 0.02-1.0 mg/mL.
Clause 9. The method according to clause 8, characterized in that in the mixture of the oregano essential oil and the diluted boar semen, the concentration of the oregano essential oil is 0.06- 0.08 mg/mL.
Clause 10. The method according to any one of clauses 4-9, characterized in that the oregano essential oil is added 1n the form of an oregano essential oil aqueous solution.
Claims (10)
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210984499.9A CN115245161A (en) | 2022-08-17 | 2022-08-17 | Oregano oil-based long-acting diluent for normal-temperature preservation of boar semen |
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| NL2034272A true NL2034272A (en) | 2024-02-27 |
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| Application Number | Title | Priority Date | Filing Date |
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| NL2034272A NL2034272A (en) | 2022-08-17 | 2023-03-06 | Long-acting diluent for boar semen preservation at normal temperature based on oregano essential oil |
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| CN (1) | CN115245161A (en) |
| NL (1) | NL2034272A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115251042B (en) * | 2022-09-08 | 2023-06-02 | 安徽科技学院 | Poultry semen dilution buffer solution and preparation method thereof and poultry semen cooling method |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3636609B2 (en) * | 1999-02-24 | 2005-04-06 | 日本全薬工業株式会社 | Pig semen preservation solution and method of use thereof |
| KR100903581B1 (en) * | 2008-07-10 | 2009-06-22 | 주식회사 노아바이오텍 | Porcine liquid semen diluent for long term preservation |
| ES2787200T3 (en) * | 2014-07-14 | 2020-10-15 | Politecnico Colombiano Jaime Isaza Cadavid | Diluent for the preservation of semen |
| CN111053080A (en) * | 2019-11-01 | 2020-04-24 | 中国农业大学 | Semen diluent for improving semen low-temperature preservation quality and application method thereof |
| CN111657263A (en) * | 2020-05-08 | 2020-09-15 | 洛宁农本畜牧科技开发有限公司 | Animal semen diluting powder and application thereof |
| CN111685102A (en) * | 2020-05-08 | 2020-09-22 | 洛宁农本畜牧科技开发有限公司 | Sheep sperm solution diluent powder |
| CN112369409B (en) * | 2020-11-23 | 2023-08-22 | 青岛农业大学 | Basic diluent for frozen semen of pig, preparation method and application thereof |
| CN113197264A (en) * | 2021-05-20 | 2021-08-03 | 山东傲农种猪有限公司 | Additive for improving boar semen quality and application thereof |
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2022
- 2022-08-17 CN CN202210984499.9A patent/CN115245161A/en active Pending
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