CN113331178A - Application of methionine in preparation of diluent for preserving boar semen at normal temperature - Google Patents
Application of methionine in preparation of diluent for preserving boar semen at normal temperature Download PDFInfo
- Publication number
- CN113331178A CN113331178A CN202110610955.9A CN202110610955A CN113331178A CN 113331178 A CN113331178 A CN 113331178A CN 202110610955 A CN202110610955 A CN 202110610955A CN 113331178 A CN113331178 A CN 113331178A
- Authority
- CN
- China
- Prior art keywords
- diluent
- semen
- preserving
- normal temperature
- boar semen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an application of methionine in preparing a diluent for preserving boar semen at normal temperature, wherein the diluent mainly comprises methionine, glucose, EDTA disodium, sodium citrate, citric acid, sodium bicarbonate, Tris, penicillin, streptomycin and polymyxin; meanwhile, the diluent is simple in preparation method, convenient to use and convenient to popularize and apply on a large scale.
Description
Technical Field
The invention relates to the technical field of pig breeding, in particular to a formula of a diluent for preserving boar semen at normal temperature.
Background
Artificial insemination technology (AI) is a technology that collects semen of male animals by an Artificial method, and inputs the semen into a specific part of the reproductive tract of female animals after a series of treatments such as semen quality inspection, dilution, preservation and the like to replace natural mating and breeding offspring. The technology effectively changes the breeding process of livestock, improves the breeding efficiency and the breeding value of excellent breeding male livestock, accelerates the breeding process and reduces the production cost. In recent years, with the continuous popularization of artificial insemination technology, more and more intensive farms choose artificial insemination to breed in order to obtain higher conception rates, and therefore, higher requirements are also put on the quality of semen preserved in vitro. In vitro preservation (in vitro storage) is a technology for prolonging the preservation time of the boar semen by mixing the manually collected semen with the standard quality with a diluent and matching with corresponding preservation facilities and preservation temperatures. The preservation mode of the semen of the male breeding stock can be divided into freezing preservation and liquid preservation according to the preservation temperature.
The cryopreservation means that the semen is preserved in dry ice (-79 ℃) or liquid nitrogen (-196 ℃) to make the metabolic activity of the sperm static, the temperature is rapidly reduced to pass through the ice crystal period (0-60 ℃) to prevent the liquid inside and outside the cells from forming ice crystals to puncture cell membranes, so that the sperm cells form a vitrified state, and the temperature is rapidly increased to make the sperm restore normal biological activity when the semen cryopreservation is applied to the female animal delivery, thereby achieving the purpose of keeping the survival rate and the fertilization capability. However, during the sperm freezing-thawing process, a great amount of ice crystals are inevitably generated in and out of the sperm cells, and the osmotic pressure and the pH value of the semen are affected, so that the sperm die greatly. Although the pig semen cryopreservation technology has advanced to a certain extent in recent years, compared with other preservation methods, the cryopreservation method has high requirements on equipment, complex and tedious operation process, poor sperm preservation effect and low conception rate, and is still mainly applied to the aspects of experimental research, germplasm resource protection and the like.
The liquid state preservation refers to adding corresponding diluent into the boar semen to provide an energy substance and a protective agent, and slowly cooling to the normal temperature (15-25 ℃) or the low temperature (0-5 ℃) so as to reduce the metabolic activity of the sperms and reduce the accumulation of the metabolic products and the energy consumption of the sperms while keeping the semen in a fluid state. The liquid state preservation not only prolongs the in vitro survival time of the sperms and improves the utilization rate of the semen, but also keeps higher survival rate and fertilization capability of the sperms.
The wide popularization of the artificial insemination of the pigs plays an important promoting role in the breeding of excellent pig species and the production of high-quality pork. With the application of the artificial insemination technology of pigs, the preservation of the boar semen becomes a key step in the artificial insemination technology. The popularity of artificial insemination of pigs by using frozen semen is very low (less than 1% in the world, the number of sperms per insemination is not less than 50 hundred million, and the conception rate is about 70%), and the liquid state of semen is widely stored in production. More than 99% of the semen is liquid semen preserved for about 3 days, the number of the semen is not less than 25 hundred million each time when the sows in estrus are inseminated for two to three times, the insemination amount is about 100mL, the conception rate reaches 80-90%, and the farrowing rate and the litter size are close to or even higher than natural mating.
The biological properties of the pig sperm, which are low cholesterol/phospholipid ratio and high phosphatidylethanolamine and sphingomyelin content, result in a higher sensitivity of the pig sperm to low temperatures than other livestock such as cattle. The low temperature can cause the damage of the structures and functions of sperm plasma membranes, ultrastructures, cell active components and the like, so that the sperm motility, the integrity of the plasma membranes and acrosomes and the reduction of the sperm mitochondrial membrane potential are reduced, and the change can reduce the conception capacity of the preserved pig sperms. Therefore, semen is currently preserved in swine artificial insemination using a normal temperature preservation (e.g., 17 ℃).
Commercial pig semen preserving dilutions are of a wide variety, with energy substances typically added to maintain the various metabolic processes of the sperm, and Tris or HEPES buffers added to control pH. The normal temperature semen preserving diluent can keep the sperm motility at about 0.8 and 60%, but the preserving time is limited, and the development requirements of enlarging the radiation range of an improved male stock station and the utilization rate of the stock can not be met. The motility of sperm is one of the important characteristics of live sperm; the movement modes of the sperms mainly comprise 3 types of linear advancing, circular movement and in-situ swinging; the sperm moving forward straight can have normal impact ability and fertilization ability; while the sperm preserved at normal temperature (17 ℃) is in a resting state. In addition, the level of Reactive Oxygen Species (ROS), a byproduct of mitochondrial oxidative phosphorylation during storage at ambient temperatures, also has some effect on sperm fertility.
Glutathione (GSH) is the most important cytokine that regulates sperm redox homeostasis. Sperm can synthesize GSH using methionine (which is a non-polar amino acid) or cysteine. The synthesized GSH can relieve plasma membrane damage caused by ROS and maintain sperm movement. Higher methionine content was found in porcine seminal plasma, while lower cysteine and cystine content. Chinese patent CN107094755A reports a diluent formula containing various amino acids including methionine for providing energy to maintain sperm motility and vitality, but the addition of various amino acids in combination may produce toxic effect on sperm, which is more obvious as the sperm metabolic activity continues, thus failing to prolong the effective preservation time of semen.
The commercial diluent Modana can only meet the normal energy requirement during the sperm preservation period within a certain preservation time range, and cannot eliminate the influence of adverse factors such as reduced energy metabolism, increased ROS generation and the like on the sperm preservation period as the preservation time is prolonged.
Disclosure of Invention
The invention aims to provide application of methionine in preparation of a diluent for preserving boar semen at normal temperature. The prepared diluent has good normal-temperature preservation effect on the boar semen.
In order to achieve the purpose, the invention adopts the following technical scheme:
the diluent for preserving the boar semen at the normal temperature comprises 27.5-28.5 g/L of glucose, 2.35-2.55 g/L of disodium EDTA, 5.65-5.85 g/L of Tris, 6.9-7.86 g/L of sodium citrate, 2.9-3.174 g/L of citric acid, 1.0-1.15 g/L of sodium bicarbonate, 60-70 ten thousand IU/L of penicillin, 180-200 ten thousand/L of streptomycin, 1.6-2 ten thousand IU/L of polymyxin and <1mmol/L of methionine.
Preferably, the pH value of the normal-temperature preservation diluent is 7.15-7.25.
Preferably, the components of the diluent for normal-temperature storage comprise 27.5g/L glucose, 2.35g/L disodium EDTA, 5.65g/L Tris, 6.9g/L sodium citrate, 2.9g/L citric acid, 1.0g/L sodium bicarbonate, 60 ten thousand IU/L penicillin, 180 ten thousand IU/L streptomycin, 1.6 ten thousand IU/L polymyxin and 0.1mmol/L methionine.
A method for preserving pig semen at normal temperature comprises the following steps:
and mixing the normal-temperature preservation diluent with the boar semen and preserving at 15-20 ℃.
Preferably, the mixing specifically comprises the steps of: diluting the collected fresh boar semen with the normal-temperature preservation diluent, and diluting the density of the boar semen to 0.5-0.6 multiplied by 108one/mL.
Preferably, the normal-temperature preservation diluent is preheated to 37-38.5 ℃ before being mixed with fresh boar semen (for example, isothermal with fresh boar semen).
Preferably, the sperm motility of the fresh boar semen is more than 80 percent, and the sperm motility rate is more than 0.80.
Preferably, the storage time is at least 7 days.
The invention has the beneficial effects that:
the invention selects and combines methionine with glucose, EDTA disodium, Tris, sodium citrate, citric acid, sodium bicarbonate, penicillin, streptomycin and polymyxin to form a diluent formula capable of efficiently preserving the boar semen, and can supplement the nutrition consumption of the sperm, maintain the osmotic pressure of the sperm, reduce the harm of sperm metabolites and damage of oxidative stress to the structure of the sperm plasma membrane. And the addition of methionine can effectively maintain the integrity of the plasma membrane of the sperms and the impact capacity of the sperms in a longer storage time, thereby maintaining higher sperm motility. Experimental results show that the diluent can ensure that the activity of the boar semen still reaches more than 70 percent after the boar semen is stored for one week at normal temperature, and effectively solves the timeliness problem of artificial insemination of fresh semen. Meanwhile, the diluent is simple and convenient to operate and convenient to popularize and apply on a large scale when used for preserving the boar semen.
Detailed Description
The present invention will be described in further detail with reference to examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
The invention takes the semen preservation requirement of an improved breed male livestock station in the expansion of the radiation range of artificial insemination to peripheral enterprises and farmers (farms) as a starting point, combines basic components such as energy substances, buffer solution and the like, screens and optimizes antibacterial agents and amino acid additives, develops a diluent formula capable of efficiently preserving the pig semen (preserving for more than one week at normal temperature), and can better overcome the adverse effects of practical conditions such as semen collection time limit, semen deposition place difference and the like on the full play of the utilization rate of improved breed male pigs and the realization of improved breed resource sharing compared with commercial diluent (such as Modena diluent).
Example 1
Preparation of diluent for preserving boar semen at normal temperature
The diluent comprises the following components in percentage by weight: 2.75g of glucose, 0.235g of EDTA disodium, 0.565g of Tris, 0.69g of sodium citrate, 0.29g of citric acid, 0.1g of sodium bicarbonate, 6 ten thousand IU of penicillin, 18 ten thousand IU of streptomycin, 0.16 ten thousand IU of polymyxin and 0.01mmol of methionine.
Weighing each component of the diluent formula according to the dosage, fully and uniformly mixing the components with double distilled water, and then fixing the volume to 100mL to obtain the diluent for preserving the boar semen at normal temperature, wherein the pH value is about 7.2.
Glucose in the diluent has the functions of providing nutrition to supplement energy consumed by sperm survival and movement and maintaining sperm high activity, and disodium EDTA, sodium citrate, citric acid, sodium bicarbonate and Tris have the functions of providing buffering and balancing osmotic pressure (the added glucose, disodium EDTA, sodium citrate, citric acid, sodium bicarbonate and Tris are nontoxic and have pH regulation and good buffering stability). On the basis, the penicillin, the streptomycin and the polymyxin which are added according to the proportion in the diluent can effectively control the bacterial colony number in the semen and have no toxic effect on the sperms, thereby maintaining the higher survival rate of the sperms. Meanwhile, the methionine added in the diluent according to the proportion has no toxic action on sperms, is beneficial to cells to generate reduced glutathione (the reduced glutathione has the characteristics of strong oxidation resistance, aging resistance, apoptosis prevention and the like), can effectively reduce lipid peroxidation of active oxygen free Radicals (ROS), has the effects of improving the movement performance of sperms, mediating signal transmission, preventing the damage of the lipid peroxidation on sperms (for example, preventing the damage of sperms membranes), resisting oxidation and the like, and can obviously improve the fluidity of sperms membranes, thereby improving the vitality of the sperms.
Method for preserving fresh essence of (II) pig at normal temperature
Fresh essence collection and quality evaluation: collecting semen of adult healthy boars by adopting a false negative method or a hand-held method, placing the collected fresh semen in a thermos cup, and sending the semen to a laboratory within 60min for quality detection.
Taking 10 mu L of fresh semen, and counting by using a blood cell counting plate, wherein the sperm density of the normal pig semen is 2-5 hundred million/mL.
Diluting the sperm density of fresh semen to 1 hundred million/mL by Modena diluent, putting the diluted semen into a water bath kettle at 37 ℃ for incubation for 10min, dripping 6 mu L of diluted semen onto a preheated glass slide, and performing sperm motility detection by using a computer aided analysis system (CASA), wherein the fresh semen with the motility of more than 80 percent can be used for preservation.
And (3) normal temperature preservation: adding a diluent prepared in the step I of isothermal (preheating to 37 ℃) according to the volume of fresh semen (the temperature is 37 ℃) and the sperm density to ensure that the sperm density is 0.5-0.6 multiplied by 108And (4) mixing the ingredients per mL, reversing, uniformly mixing, sealing, keeping away from light and storing in an environment at 17 ℃ for more than 7 days, and repeatedly using the ingredients for artificial insemination according to specific conditions.
(III) evaluation of quality of preserved pig semen
Sperm motility, motility rate and other sperm movement parameters are measured: taking a semen sample to be detected on the same day (namely semen preserved at normal temperature) from a constant-temperature refrigerator at 17 ℃, incubating the semen sample in a water bath kettle at 37 ℃ for 10min, and then taking 10 mu L of the sample to measure the sperm motility rate and activity, the sperm linear motion rate and the sperm curvilinear motion rate by using a computer aided analysis system (CASA) under the 250 times of microscope visual field; and selecting more than 5 different visual fields during measurement, wherein the number of sperms in each visual field is more than 200, and the sperms are uniformly distributed for detection.
Sperm plasma membrane integrity test (using PI/SYBR-14 method): taking out the semen sample to be detected on the same day from a 17 ℃ constant temperature refrigerator, putting 100 mu L of the semen sample into a 1.5mL centrifuge tube, incubating for 5min in a 37 ℃ water bath kettle, adding 0.2 mu L of SYBR-14 working solution, heating for 5min in a 37 ℃ water bath after uniformly mixing, adding 0.3 mu L of PI working solution, uniformly mixing, putting 2.5 mu L of dyed semen on a glass slide, covering a cover glass, and respectively exciting by using 488nm blue light and 525nm green light to observe the complete (SYBR-14 dyeing) and damaged (PI dyeing) conditions of the plasma membrane. Randomly selecting 5 clear visual fields for observation, counting more than 200 sperms in each visual field, and taking pictures under a fluorescence microscope. The percentage of the sperms with complete plasma membranes in the semen to the total number of the sperms is the plasma membrane complete rate of the sperms; the sperm is the only cell which can play a role in vitro, and the maintenance of the integrity of the plasma membrane is the basis for maintaining the normal operation of the internal system and is the fundamental guarantee for the sperm to finish fertilization.
Sperm mitochondrial membrane potential assay (detection of mitochondrial membrane potential of sperm using fluorescent dye JC-1): taking 50 mu L semen sample, centrifuging for 5min at 800g, removing supernatant, collecting sperm cell precipitate, and washing twice with Modena diluent; adding 200 mu L JC-1 dyeing working solution, gently blowing and uniformly mixing by using a liquid transfer gun, and incubating at the constant temperature of 37 ℃ for 20-30 min; after the incubation is finished, centrifuging for 5min at 800g, collecting spermocyte precipitates, and washing for 2-3 times by using precooled JC-1 staining buffer solution (1 x); resuspending with JC-1 staining buffer (1X), placing on ice, detecting twenty thousand sperms (excitation wavelength 525/488nm, emission wavelength 590/525nm) by using a multifunctional microplate reader, wherein the sperms emitting orange-red fluorescence are the sperms with higher mitochondrial membrane potential, the sperms emitting green fluorescence are the sperms with lower mitochondrial membrane potential, and the higher membrane potential represents the higher energy-producing activity of mitochondria.
Example for preserving boar semen at normal temperature
Adult and healthy 10 boars are selected in the Yangling demonstration area of Shaanxi province, semen is collected by a hand-held method, and the apparent motility rate, the vitality and the density of the semen are detected at 37 ℃, so that the quality of the semen collected from each pig is evaluated, and the results are shown in Table 1. The result shows that the density of the collected fresh sperms of the pig is 3-4 multiplied by 108Within the range of one/mL, the apparent activity rates reach the highest values, the activity rates are more than 0.80, the average value is about 0.9, and the activity is more than 80%.
Preserving porcine semen for one week by the preservation method (mixing fresh semen with diluent to make the fresh semenSperm density of 0.5 × 108one/mL, after mixing, the mixture was stored in a constant temperature refrigerator at 17 ℃), and the quality of the fresh essence was compared with that of the fresh essence by using Modena diluent as an experimental control.
The results show (table 1) that the sperm motility rate of about 0.9, the motility of more than 70 percent and the plasma membrane integrity rate of more than 70 percent after the pig semen is preserved for one week at normal temperature by using the diluent of the invention.
TABLE 1 Effect of the dilution on Normal temperature preservation of porcine semen
| Control group (Modena diluent) | The diluent of the invention | |
| Quantity (head) | 10 | 10 |
| Fresh precision (× 10)8one/mL) | 3.55±0.36 | 3.55±0.36 |
| Fresh essence survival rate | 0.90±0.09 | 0.90±0.09 |
| Survival rate after preservation | 0.87±0.09 | 0.91±0.055 |
| Viability after preservation (%) | 64.86±1.52 | 72.38±1.18 |
| Plasma membrane integrity after storage (%) | 66.62±0.16 | 73.7±0.19 |
| Mitochondrial membrane potential after storage | 3.89±0.25 | 4.55±0.35 |
The result shows that compared with Modena diluent, the diluent disclosed by the invention can effectively eliminate the influence of adverse factors on the sperm during preservation, and provides a reliable basis for prolonging the preservation time of the semen. Meanwhile, the diluent avoids the toxic action on sperms caused by improper addition of amino acid.
In conclusion, the diluent formula disclosed by the invention has a good effect on normal-temperature preservation of the pig semen, not only is the semen preserved for a long time, but also the sperm motility and vitality can be effectively maintained, so that the sperm motility of the pig can still reach more than 70% after the fresh pig semen is stored for one week at normal temperature, the requirement of an improved breed male stock station on expanding the radiation range of artificial insemination is completely met, and the production efficiency of the artificial insemination is favorably improved. Meanwhile, the diluent is simple in formula and easy to prepare, is convenient for large-scale popularization and application, is beneficial to improving the utilization rate of the fine breed boars and obtaining high conception rate, and realizes the real fine breed resource sharing.
Claims (8)
1. The diluent for preserving the boar semen at normal temperature is characterized in that: the diluent for normal-temperature preservation comprises 27.5-28.5 g/L glucose, 2.35-2.55 g/L disodium EDTA, 5.65-5.85 g/L Tris, 6.9-7.86 g/L sodium citrate, 2.9-3.174 g/L citric acid, 1.0-1.15 g/L sodium bicarbonate, 60-70 ten thousand IU/L penicillin, 180-200 ten thousand IU/L streptomycin, 1.6-2 ten thousand IU/L polymyxin and <1mM methionine.
2. The diluent for preserving the boar semen at the normal temperature as claimed in claim 1, wherein: the pH value of the normal-temperature storage diluent is 7.15-7.25.
3. The diluent for preserving the boar semen at the normal temperature as claimed in claim 1, wherein: the diluent for normal-temperature preservation comprises 27.5g/L glucose, 2.35g/L disodium EDTA, 5.65g/L Tris, 6.9g/L sodium citrate, 2.9g/L citric acid, 1.0g/L sodium bicarbonate, 60 ten thousand IU/L penicillin, 180 ten thousand IU/L streptomycin, 1.6 ten thousand IU/L polymyxin and 0.1mmol/L methionine.
4. A method for preserving boar semen at normal temperature is characterized in that: the method comprises the following steps:
mixing the diluent with the boar semen and then preserving at 15-20 ℃; the diluent comprises 27.5-28.5 g/L glucose, 2.35-2.55 g/L disodium EDTA, 5.65-5.85 g/L Tris, 6.9-7.86 g/L sodium citrate, 2.9-3.174 g/L citric acid, 1.0-1.15 g/L sodium bicarbonate, 60-70 ten thousand IU/L penicillin, 180-200 ten thousand IU/L streptomycin, 1.6-2 ten thousand IU/L polymyxin and <1mmol/L methionine.
5. The method for preserving boar semen at normal temperature as claimed in claim 4, wherein the method comprises the following steps: the mixing specifically comprises the following steps: diluting the collected fresh boar semen with the diluent, and diluting the density of the boar semen to 0.5-0.6 multiplied by 108one/mL.
6. The method for preserving boar semen at normal temperature as claimed in claim 4, wherein the method comprises the following steps: the diluent is preheated to 37-38.5 ℃ before being mixed with the boar semen.
7. The method for preserving boar semen at normal temperature as claimed in claim 4, wherein the method comprises the following steps: the sperm motility of the pig semen is more than 80%, and the sperm motility rate is more than 0.80.
8. The method for preserving boar semen at normal temperature as claimed in claim 4, wherein the method comprises the following steps: the storage time is at least 7 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110610955.9A CN113331178B (en) | 2021-06-01 | 2021-06-01 | Application of methionine in preparing normal-temperature preservation diluent for boar semen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110610955.9A CN113331178B (en) | 2021-06-01 | 2021-06-01 | Application of methionine in preparing normal-temperature preservation diluent for boar semen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113331178A true CN113331178A (en) | 2021-09-03 |
| CN113331178B CN113331178B (en) | 2023-04-21 |
Family
ID=77474376
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110610955.9A Active CN113331178B (en) | 2021-06-01 | 2021-06-01 | Application of methionine in preparing normal-temperature preservation diluent for boar semen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113331178B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101622986A (en) * | 2009-08-10 | 2010-01-13 | 浙江大学 | Glutathione-contained boar semen cryopreservation liquid and cryopreservation method thereof |
| CN103299987A (en) * | 2013-05-29 | 2013-09-18 | 西北农林科技大学 | Diluent for normal temperature preservation of boar semens and preparation method thereof |
| CN107094755A (en) * | 2017-06-23 | 2017-08-29 | 四川农业大学 | It is a kind of to improve freeze proof diluent of nutrition that sperm in vitro is preserved and preparation method thereof |
-
2021
- 2021-06-01 CN CN202110610955.9A patent/CN113331178B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101622986A (en) * | 2009-08-10 | 2010-01-13 | 浙江大学 | Glutathione-contained boar semen cryopreservation liquid and cryopreservation method thereof |
| CN103299987A (en) * | 2013-05-29 | 2013-09-18 | 西北农林科技大学 | Diluent for normal temperature preservation of boar semens and preparation method thereof |
| CN107094755A (en) * | 2017-06-23 | 2017-08-29 | 四川农业大学 | It is a kind of to improve freeze proof diluent of nutrition that sperm in vitro is preserved and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 朱振东: "猪精子能力代谢的调控机理研究", 《中国优秀博硕士学位论文全文库（博士）_农业科技辑》 * |
| 王怀禹: "《畜禽繁殖与改良技术》", 31 October 2015, 西安交通大学出版社 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113331178B (en) | 2023-04-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Naijian et al. | Effects of different concentrations of BHT on microscopic and oxidative parameters of Mahabadi goat semen following the freeze–thaw process | |
| Yildiz et al. | Effect of cholesterol-loaded cyclodextrin on cryosurvival and fertility of cryopreserved carp (Cyprinus carpio) sperm | |
| Lahnsteiner et al. | Effects of media, fertilization technique, extender, straw volume, and sperm to egg ratio on hatchability of cyprinid embryos, using cryopreserved semen | |
| US20120264105A1 (en) | Composition for Preserving Reproductive Cells and Method of Using | |
| CN114208814A (en) | Diluent for frozen pig semen and its preparing process and application | |
| Naijian et al. | Effects of various concentrations of BSA on microscopic and oxidative parameters of Mahabadi goat semen following the freeze–thaw process | |
| US20230276790A1 (en) | Hypothermic preserving diluent, hypothermic preserving method of plectropomus leopardus semen and application thereof | |
| CN103299987A (en) | Diluent for normal temperature preservation of boar semens and preparation method thereof | |
| Shaoyong et al. | Evaluation of ε-polylysine as antimicrobial alternative for liquid-stored boar semen | |
| Inanc et al. | Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol | |
| Vongpralub et al. | Comparison of effects of different antioxidants supplemented to long-term extender on boar semen quality following storage at 17ºC | |
| CN109907038A (en) | A kind of non-animal derived ingredient frozen cattle semens dilution | |
| Liu et al. | Effect of L-proline on sperm quality during cryopreservation of boar semen | |
| CN110012901B (en) | Diluting preparation for preserving boar semen at normal temperature | |
| CN113331178B (en) | Application of methionine in preparing normal-temperature preservation diluent for boar semen | |
| CN115119833B (en) | Semen cryopreservation diluent and application thereof | |
| CN106818709A (en) | Cattle freezing seminal fluid dilution preparation method | |
| Chowdhury et al. | Seminal vesicle and testis secretions in Heteropneustes fossilis (Bloch): composition and effects on sperm motility and fertilization | |
| CN113331179A (en) | Chinese local pig semen cryopreservation agent and application thereof | |
| CN113331175B (en) | Method for improving normal-temperature preservation effect of boar semen by using L-arginine | |
| CN113273566A (en) | Application of glycine as effective component in boar semen normal-temperature preservation solution | |
| Sorongbe et al. | Soybean milk extender improves sperm functional and oxidative stress parameters of goat sperm during slow and rapid freezing | |
| CN110079495B (en) | Frozen bovine sperm activating liquid | |
| CN117958253A (en) | Application of limonin, inositol and/or L-proline in pig semen cryopreservation | |
| CN115119828B (en) | Diluent for refrigerating and preserving canine sperms and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |