CN107232184B - Diluent for preserving semen of black pig in Guanzhong at normal temperature - Google Patents
Diluent for preserving semen of black pig in Guanzhong at normal temperature Download PDFInfo
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- CN107232184B CN107232184B CN201710619760.4A CN201710619760A CN107232184B CN 107232184 B CN107232184 B CN 107232184B CN 201710619760 A CN201710619760 A CN 201710619760A CN 107232184 B CN107232184 B CN 107232184B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
Abstract
The invention discloses a diluent for preserving semen of black pigs at normal temperature, which mainly comprises glucose, sodium citrate, ethylene diamine tetraacetic acid, sodium bicarbonate, potassium chloride, kojic acid, epsilon-polylysine and the like. The invention can store the semen of the Guanzhong black pig in a liquid state at normal temperature for 1-5 days and meet the requirements of artificial insemination. Kojic acid is added into the diluent, so that the antioxidant protection effect on sperms is good, the using amount of antibiotics in pig raising production is reduced, and the problem of bacterial drug resistance can be effectively relieved, so that the problem of pig semen preservation of artificial insemination technology in the large-scale breeding process of Guanzhong black pigs is solved, the breeding of boars is reduced, and the utilization rate of excellent boar semen is improved. The diluent has the advantages of simple preparation method, simple operation, low production cost and convenient large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a normal-temperature semen storage diluent formula for semen deposition in deep parts of Guanzhong black pigs.
Background
The Guanzhong black pigs are the product varieties of the geographical signs of the ministry of agriculture and are named after being produced and widely distributed in the plain of the Shanxi Guanzhong. The Guanzhong black pig is a meat and fat dual-purpose variety obtained by hybridizing and breeding a Bake summer pig, a Gansu Jingchuan eight-eyebrow pig and a Sichuan Nenjiang pig, completes identification in the early 80 years, and has excellent germplasm characteristics of high reproductive capacity, good meat quality, coarse feeding resistance, strong stress resistance and the like.
In the process of promoting the large-scale breeding of the Guanzhong black pigs, the use efficiency of the semen of the high-quality breeding boars can be improved by using the deep insemination technology, the breeding amount of the boars is reduced, the production cost is reduced, and the economic benefit of a pig farm is improved. However, there are still some significant problems in the use of this artificial insemination technique. For example, the boar semen is inevitably polluted by microorganisms in the processes of collection, dilution, subpackaging and later-period preservation, and because the microorganisms have great harm to the preservation of the semen, the growth of bacteria in the semen is mainly inhibited by adding antibiotics into the diluent so as to reduce the harm of the bacteria to the semen. However, in recent years, the unscientific use of antibiotics has led to the development of bacterial resistance, and the continued addition of antibiotics to dilutions may lead to the infection of sows by semen-directed resistant bacteria, which may lead to a reduction in reproductive performance of sows after insemination using semen contaminated with these bacteria, and may increase treatment costs for sows when these bacteria are causing disease again. In the current commercial operation of the pig semen, the pig semen is very easily subjected to oxidative stress due to a large amount of unsaturated fatty acid contained in a membrane in the process of storage and transportation, which has extremely adverse effects on the structural integrity and the normal function of the semen and reduces the reproductive performance of the sows after insemination. Therefore, in order to adapt to the development of the artificial insemination technology of the pigs, promote the popularization and the use of the deep insemination technology of the black pigs in Guanzhong, improve the semen quality and the breeding performance of sows, which are preserved at normal temperature, reduce the production cost of a pig farm and improve the production benefit, the diluent suitable for the preservation of the black pig semen in Guanzhong at normal temperature is urgently needed to be developed under the current situation.
The product diluent has more brands and mixed fish and dragon, and has the following problems in practical application:
1) the boar semen normal temperature preservation diluent powder widely used in the market at present is mainly of foreign brands, and has few products independently researched and developed at home, and the diluent powder is not necessarily suitable for the dilution preservation of the Guanzhong black boar semen.
2) Antibiotics are mainly added into the commercial diluent for sterilization and bacteriostasis, however, in recent years, the problems of antibiotic residues and bacterial drug resistance are increasingly prominent, and the problem is to be solved.
3) The sperm is easy to generate oxidative stress phenomenon in the normal temperature preservation process, and the quality of the semen is affected, so that the reproductive performance of the sow is reduced.
Disclosure of Invention
The invention aims to provide a diluent for preserving semen of Guanzhong black pigs at normal temperature.
In order to achieve the purpose, the invention adopts the following technical scheme.
The diluent for normal-temperature preservation comprises the components of double distilled water, 35-40 g/L glucose, 5.5-6.5 g/L sodium citrate, 5.5-6.5 g/L ethylene diamine tetraacetic acid, 1.0-1.25 g/L sodium bicarbonate, 0.5-1.0 g/L potassium chloride, 0.02-0.06 g/L kojic acid and 0.04-0.16 g/L epsilon-polylysine.
Preferably, the diluent is stored at normal temperature, the osmotic pressure is adjusted to 240 mosm-380 mosm, and the pH value is 6.4-7.2.
Preferably, the diluent for normal-temperature storage comprises double distilled water, 37.15g/L glucose, 6.0g/L sodium citrate, 6.0g/L ethylenediaminetetraacetic acid, 1.25g/L sodium bicarbonate, 0.75g/L potassium chloride, 0.02-0.06 g/L kojic acid and 0.04-0.16 g/L epsilon-polylysine. The usage amount of kojic acid and epsilon-polylysine is related to the survival rate of preserved sperms, the integrity of acrosomes and the integrity of plasma membranes.
More preferably, the components of the diluent for normal-temperature storage comprise double distilled water, 37.15g/L glucose, 6.0g/L sodium citrate, 6.0g/L ethylenediaminetetraacetic acid, 1.25g/L sodium bicarbonate, 0.75g/L potassium chloride, 0.04g/L kojic acid and 0.08g/L epsilon-polylysine.
Preferably, after the diluent stored at the normal temperature is mixed with the semen, the density of the semen is diluted to 2 x 107-3 x 107one/mL, and the preservation time is at least 120 hours.
Preferably, the normal-temperature preservation diluent is mixed with the semen and then preserved at the temperature of 15-20 ℃ (the optimal preservation temperature is 17 ℃).
The invention has the beneficial effects that:
the normal-temperature semen preservation diluent for the black pig in Guanzhong takes the problem of semen preservation of the black pig in Guanzhong as a starting point, and develops a diluent formula capable of efficiently preserving semen at normal temperature by taking glucose-sodium citrate-sodium bicarbonate-potassium chloride as a basic component. The diluent has a good normal-temperature liquid preservation effect on semen of black pigs in Guanzhong, solves the problem of pig semen preservation of artificial insemination technology in the large-scale culture process of black pigs in Guanzhong, reduces the breeding of boars, and improves the utilization rate of the fine boar semen; meanwhile, the diluent is simple in preparation method, low in production cost, easy to use and convenient for large-scale popularization and application.
Detailed Description
The present invention will be described in detail with reference to examples.
Formula of normal-temperature preservation diluent for semen in deep insemination of Guanzhong black pigs
The invention takes the semen preservation problem in the deep insemination of the black pig in Guanzhong as a starting point, a diluent formula capable of efficiently preserving the semen at normal temperature (15-20 ℃) is developed by taking glucose-sodium citrate-sodium bicarbonate-potassium chloride as a basic component and adding cationic antimicrobial peptide epsilon-polylysine and antioxidant kojic acid, the kojic acid added in the diluent has good antioxidant protection effect and certain antibacterial capacity on the semen, and the cationic antimicrobial peptide epsilon-polylysine is further combined to replace antibiotics, so that the problem of easy oxidative stress of the semen in the normal-temperature preservation process of the pig semen is effectively relieved, the using amount of the antibiotics in pig raising production is reduced, the problem of bacterial drug resistance can be effectively relieved, and the use of the antibiotics and the generation of drug-resistant bacteria are greatly reduced. The invention is beneficial to expanding the semen utilization rate of the boars of the excellent breeds, ensuring the health of the sows, improving the conception rate of deep insemination and realizing the high-efficiency and low-cost deep insemination.
The invention discloses a normal-temperature preservation diluent for semen of deep insemination of Guanzhong black pigs, which is prepared by adding 0.02-0.06 g/L kojic acid and 0.04-0.16 g/L epsilon-polylysine on the basis of glucose, sodium citrate, ethylene diamine tetraacetic acid, sodium bicarbonate and potassium chloride, mixing with double distilled water, and adjusting osmotic pressure to 240-380 mosm and pH to 6.4-7.2.
Glucose has the function of providing nutrition to supplement the energy consumed by the sperm for survival and movement, maintaining the osmotic pressure of the semen. The sodium citrate and the sodium bicarbonate have no toxicity, pH regulation performance and good buffering stability, and the sodium citrate and the sodium bicarbonate which are added in proportion in the diluent achieve ideal effects of buffering and osmotic pressure balancing. The ethylene diamine tetraacetic acid is a nontoxic organic chelating agent, can be chelated with metal ions, and reduces the damage of the metal ions to sperms. The addition of potassium chloride plays an important role in maintaining the normal operation of the sperm cell sodium potassium pump. The invention is characterized in that: antibiotics are not added, so that the proliferation of bacteria in semen can be effectively inhibited, the in-vitro storage of sperms is guaranteed, the use and the residue of the antibiotics in the livestock production are reduced, and the probability of drug-resistant bacteria is reduced. Secondly, the compound has the characteristics of strong oxidation resistance, bacterial growth inhibition, metal ion chelation and the like, can effectively reduce the lipid peroxidation of harmful substance active oxygen free Radicals (ROS), and improves the total oxidation resistance (T-AOC) of sperms. Therefore, the sperm cell has the effects of improving the motility rate and the movement performance of the sperm, the integrity of a plasma membrane, the integrity of a acrosome and the integrity of mitochondria and the like.
The formula of the normal-temperature semen preservation diluent for the deep insemination of the Guanzhong black pigs is further characterized by being simple in operation, economical and practical, all the components are fully mixed in proportion and then are subjected to isothermal dilution with fresh semen and sealed preservation, and the semen is isolated from air, so that the semen is prevented from being damaged by the external environment. The survival rate can still reach more than 60 percent after the pig is stored for 120 hours at normal temperature, and the requirement of a pig farm on deep insemination is completely met.
The diluent for preserving semen of deep insemination of Guanzhong black pigs at normal temperature comprises the following components in optimal content: 37.15g of glucose, 6.0g of sodium citrate, 6.0g of ethylenediamine tetraacetic acid, 1.25g of sodium bicarbonate, 0.75g of potassium chloride, 0.04g of kojic acid and 0.08g of epsilon-polylysine. Mixing the above components with 1L double distilled water to obtain semen normal temperature preservation diluent for deep insemination of Guanzhong black pig. The pH value of the diluent is 6.4-6.8.
Method for preserving fresh essence of black pig in Guanzhong
Fresh essence collection and quality evaluation: the method comprises the steps of collecting semen of an adult healthy boar by a double-layer glove hand-held method, carrying out quality evaluation on the motility rate and the density of the semen at 37 ℃, detecting the motility rate and the movement performance of the semen under a computer aided analysis system (CASA), diluting and storing a sample with the motility rate higher than 80%, and simultaneously detecting and displaying the semen density when the sperm motility rate is detected by the computer aided analysis system (CASA).
The normal temperature preservation method comprises the following steps: adding isothermal normal temperature preservation diluent (in the optimum formula content) of semen for deep insemination of the Guanzhong black pig according to the volume and density of the collected fresh semen (at the temperature of 37 ℃) to ensure that the semen density is 2.5 multiplied by 107The semen is put into an 80mL semen bottle after being gently shaken and uniformly mixed, the semen is slowly cooled for 1-2 h to room temperature according to the air temperature, and then the semen is transferred to a constant temperature refrigerator at 17 ℃ for storage for 120h, and the semen can be used for production according to the production requirement in the periodDeep insemination of pigs.
(III) evaluation of quality of preserved semen of black pig in Guanzhong
And (3) sperm motility rate determination: before insemination, 10 mu L of sample is taken to detect the sperm motility rate and the motility performance under a computer aided analysis system (CASA). The sperm motion performance parameters mainly comprise the sperm linear motion speed, the curve motion speed and the sperm side swing amplitude.
And (3) detecting the integrity of the sperm plasma membrane: the hypotonic swelling test (HOST) method is used for detection. Adjusting sperm density to 2 × 10 with fructose-sodium citrate hypotonic solution6And (4) incubating each sperm/mL at 37 ℃ for 30min, taking 10 mu L of semen, immediately calculating the sperm swelling condition under a 400-time phase contrast microscope, and counting the sperm plasma membrane integrity rate.
And (3) detecting the integrity rate of the sperm acrosome: 10 mu L of sample is taken to prepare a smear, after the combined staining of fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) which is a fluorescent dye, the acrosome integrity of every 200 sperms is randomly calculated under a fluorescence microscope, and the calculation is carried out for more than 3 times.
Detection of sperm mitochondrial membrane integrity: 10 mu L of sample is taken to prepare a smear, and after the smear is jointly dyed by using a fluorescent dye rhodamine-propidium iodide (Rh123-PI), the integrity of each 200 sperm mitochondrial membranes is randomly calculated under a fluorescent microscope, and the calculation is carried out for more than 3 times.
(IV) concrete examples of experiments
Semen of 3 adult black breeding boars (1.2-2 years old) in healthy customs is collected by a hand-held method, and the survival rate and the density of the semen are detected by a computer aided analysis system (CASA). The semen was stored for the indicated time using the above dilution and procedure, and the traditional BTS solution was used as the experimental control. The quality of the semen of each boar was assessed. The result shows that the density of the collected boar fresh essence sample is 6.2-12.5 multiplied by 109The average density is 0.89, the activity rate is 82.69-91.78%, and the average value is 86.75%. After being stored for 120 hours at normal temperature, the survival rate is changed within the range of 65.58-67.26 percent, the average value is 66.42 percent, the average value of the acrosome integrity rate is 79.59 percent, and the average value of the plasma membrane integrity rate is 56.77 percent. The data results are shown in table 1.
The diluent disclosed by the invention has the main functions of supplementing nutrient substances for sperms, maintaining osmotic pressure of the sperms, reducing the damage of bacteria in the sperms to the sperms, reducing the damage of metabolites of the sperms to the sperms and reducing the damage of active oxygen in the sperms to a sperm membrane structure. Experiments prove that the diluent formula disclosed by the invention has a good effect on normal-temperature preservation of semen of black pigs in Guanzhong (for example, 0.08g/L epsilon-polylysine and 0.04g/L kojic acid cannot influence the osmotic pressure of the semen, the bacterial quantity in the semen can be effectively controlled, and the sperm motility rate reaches more than 60% after 120 hours of normal-temperature preservation and meets the production requirement), can effectively inhibit the growth of bacteria in the semen, maintain the sperm motility rate and the acrosome integrity rate, reduce the sperm aberration rate, effectively improve the conception rate of artificial insemination of pigs and the in vitro insemination efficiency, and improve the production efficiency of artificial insemination. Meanwhile, kojic acid and epsilon-polylysine in the diluent are safe and harmless and are safe substances without drug residues.
TABLE 1 Effect of the dilution according to the invention on the liquid preservation of Guanzhong Black pig semen
In addition, the invention has two functions of oxidation resistance and antibiosis, and both kojic acid and epsilon-polylysine are applied in food addition, thereby being pollution-free and low in cost, and reducing the use of antibiotics in production.
Claims (6)
1. The utility model provides a semen normal temperature preservation diluent of black pig in guan Zhong which characterized in that: the normal-temperature preservation diluent consists of the following components: the biological agent comprises the following components of double distilled water, 35-40 g/L glucose, 5.5-6.5 g/L sodium citrate, 5.5-6.5 g/L ethylene diamine tetraacetic acid, 1.0-1.25 g/L sodium bicarbonate, 0.75-1.0 g/L potassium chloride, 0.02-0.06 g/L kojic acid and 0.04-0.16 g/L epsilon-polylysine.
2. The normal-temperature semen preservation diluent for the Guanzhong black pigs, which is disclosed by claim 1, is characterized in that: the osmotic pressure of the diluent stored at normal temperature is 240-380 mosm, and the pH value is 6.4-7.2.
3. The normal-temperature semen preservation diluent for the Guanzhong black pigs, which is disclosed by claim 1, is characterized in that: the normal-temperature preservation diluent consists of the following components: double distilled water, 37.15g/L glucose, 6.0g/L sodium citrate, 6.0g/L ethylene diamine tetraacetic acid, 1.25g/L sodium bicarbonate, 0.75g/L potassium chloride, 0.02-0.06 g/L kojic acid and 0.04-0.16 g/L epsilon-polylysine.
4. The diluent for preserving semen of the black pig at normal temperature according to claim 1 or the above, which is characterized in that: the normal-temperature preservation diluent consists of the following components: double distilled water, 37.15g/L glucose, 6.0g/L sodium citrate, 6.0g/L ethylene diamine tetraacetic acid, 1.25g/L sodium bicarbonate, 0.75g/L potassium chloride, 0.04g/L kojic acid and 0.08g/L epsilon-polylysine.
5. The normal-temperature semen preservation diluent for the Guanzhong black pigs, which is disclosed by claim 1, is characterized in that: after the diluent stored at normal temperature is mixed with the semen, the density of the semen is diluted to 2 multiplied by 107～3×107one/mL, and the preservation time is at least 120 hours.
6. The normal-temperature semen preservation diluent for the Guanzhong black pigs, which is disclosed by claim 1, is characterized in that: and mixing the normal-temperature preservation diluent with the semen and then preserving at 15-20 ℃.
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| CN110326612A (en) * | 2019-08-21 | 2019-10-15 | 西北农林科技大学 | Anti-oxidant dilution of a kind of porcine semen at normal temperature preservation and preparation method thereof |
| CN113317312B (en) * | 2021-06-01 | 2022-07-12 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
Citations (3)
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| CN1712519A (en) * | 2004-06-25 | 2005-12-28 | 华中农业大学 | Liquid for preserving pig semen |
| WO2008117026A1 (en) * | 2007-03-23 | 2008-10-02 | The Royal Veterinary College | Method for enhancing sperm survival |
| CN101856016A (en) * | 2010-06-19 | 2010-10-13 | 张尤嘉 | Diluent for preserving porcine semen at normal temperature |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712519A (en) * | 2004-06-25 | 2005-12-28 | 华中农业大学 | Liquid for preserving pig semen |
| WO2008117026A1 (en) * | 2007-03-23 | 2008-10-02 | The Royal Veterinary College | Method for enhancing sperm survival |
| CN101856016A (en) * | 2010-06-19 | 2010-10-13 | 张尤嘉 | Diluent for preserving porcine semen at normal temperature |
Non-Patent Citations (1)
| Title |
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| 《Σ-聚赖氨酸对公猪***中常见细菌的体外抑制效果研究》;谢景兴等;《养猪》;20170610(第3期);第51-53页 * |
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