CN117296830A

CN117296830A - Boar semen diluent and preparation method and application thereof

Info

Publication number: CN117296830A
Application number: CN202311244747.7A
Authority: CN
Inventors: 杨明; 刘直泓; 刘峻荣; 欧稳
Original assignee: Guangdong Pig Fairy Breeding Technology Co ltd; Zhongkai University of Agriculture and Engineering
Current assignee: Guangdong Pig Fairy Breeding Technology Co ltd; Zhongkai University of Agriculture and Engineering
Priority date: 2023-09-25
Filing date: 2023-09-25
Publication date: 2023-12-29

Abstract

A boar semen diluent, a preparation method and application thereof, wherein the diluent comprises the following components in parts by mass: glucose, fructose, lactose, trehalose, sodium bicarbonate, sodium citrate, tris, EDTA-2Na, potassium chloride and antibiotic compositions; the antibiotic composition is a combination of penicillin and streptomycin, or is gentamicin, or is enrofloxacin. The preparation method is used for preparing the boar semen diluent. The most suitable for the semen can lead the vitality of the sperms to reach more than 0.6 at the temperature of 17 ℃ for 11 days, the deformity rate to be less than 18 percent and the most suitable for 11 days, and the cost required by the antibiotic is about 0.3 yuan per liter, thereby realizing the most suitable components, content and compatibility among the components of the semen diluent and solving the problem that the vitality, deformity rate, storage time and cost comprehensive performance of the sperms can not be simultaneously combined.

Description

Boar semen diluent and preparation method and application thereof

Technical Field

The invention relates to the technical field of livestock breeding, in particular to a boar semen diluent, a preparation method and application thereof.

Background

The artificial insemination technology of pigs comprises the technical steps of detecting semen quality, diluting, controlling temperature and the like of boars, and then manually injecting semen into the reproductive tract of sows in estrus, so as to realize the purpose of conception. Artificial insemination is a high-efficiency biotechnology, can improve the yield of sows and the utilization efficiency of breeding pigs, and semen dilution is one of the most important links. The preparation of the semen diluent is mainly used for supplementing energy required by the in-vitro movement of sperms, regulating the pH value of the semen at the in-vitro constant temperature, maintaining the osmotic pressure of the semen constant and providing a basic environment for the survival of the sperms at the in-vitro constant temperature. Mixing and diluting the semen diluent and the semen of the pigs according to a certain proportion to obtain standard commercial semen for artificial insemination and breeding;

the existing dilution powder put into production in farms is mostly imported brand dilution powder abroad, because the self-matched dilution powder and the domestic dilution powder have great differences from the imported dilution powder in preservation effect and insemination effect, when most boar stations produce semen, the imported dilution powder with high price and good semen preservation effect is preferentially selected for semen production, so that the semen can keep better vigor after long-distance transportation reaches a destination, the insemination quality of sow mating is improved, and better income is obtained. The cost of the diluent powder is a large proportion of the whole artificial insemination cost, so that the development of the economic and efficient domestic boar semen diluent powder can reduce the operation cost of a boar station and promote the rapid development of the artificial insemination industry of pigs in China, which is significant. The formulation of the semen diluent at present needs to be further optimized, such as the influence of the selected components, the content and the compatibility of the components of the semen diluent on the sperm motility, the deformity rate, the preservation time and the cost of the boar semen diluent. How to prepare a diluent with high comprehensive performance is the current research focus, the research and development and optimization of the semen diluent can effectively improve the quality of semen, inhibit the growth and propagation of bacteria in the semen, prolong the semen preservation time and promote the application and popularization of the artificial insemination technology.

Disclosure of Invention

The invention aims to provide a boar semen diluent, which is prepared by adding penicillin and streptomycin combination, gentamicin or enrofloxacin into a diluent consisting of glucose, fructose, lactose, trehalose, sodium bicarbonate, sodium citrate, tris, EDTA-2Na and potassium chloride.

The invention also provides a preparation method of the boar semen diluent, which is used for preparing the diluent.

The invention also provides application of the diluent in preparation of the boar semen diluent with low antibacterial cost.

The invention also provides application of the diluent in preparation of boar semen diluent with high preservation effect.

To achieve the purpose, the invention adopts the following technical scheme:

the boar semen diluent comprises the following components in parts by mass: 25-35 parts of glucose, 15-25 parts of fructose, 3-7 parts of lactose, 1-2 parts of trehalose, 1-2 parts of sodium bicarbonate, 5-7 parts of sodium citrate, 5-7 parts of Tris, 2-3 parts of EDTA-2Na, 0.1-1 part of potassium chloride and an antibiotic composition;

the antibiotic composition is a combination of 0.1-0.5 part of penicillin and 0.2-0.7 part of streptomycin, or 0.05-0.15 part of gentamicin, or 0.05-0.15 part of enrofloxacin.

The preparation method of the boar semen diluent is used for preparing the boar semen diluent and comprises the following steps:

(1) Weighing the components of the dilution of boar semen according to the formula dosage, and adding the weighed reagent into a sterilized container;

(2) Weighing 1L of ultrapure water, pouring into the container in the step (1), and stirring until the components are fully dissolved;

(3) Adding citric acid to adjust the pH value of the diluent to be between 6.4 and 6.5;

(4) After the dissolution is completed, pouring the mixture into a clean container, sealing the container, and storing the container in a refrigerator at 4 ℃.

The application of the diluent in preparation of the boar semen diluent with low antibacterial cost is disclosed, wherein the diluent is the boar semen diluent.

The application of the diluent in preparing the boar semen diluent with high preservation effect is disclosed.

The technical scheme provided by the invention can comprise the following beneficial effects:

the scheme provides a boar semen diluent, which is prepared by adding penicillin and streptomycin combination and gentamicin or enrofloxacin into a diluent composed of glucose, fructose, lactose, trehalose, sodium bicarbonate, sodium citrate, tris, EDTA-2Na and potassium chloride, wherein the diluent can optimally enable the activity of sperms to reach more than 0.6 at the temperature of 17 ℃ for 11 days, the deformity rate is less than 18% and optimally maintained to 11 days, the cost required by the antibiotic is at least about 0.3 yuan per liter, the optimal compatibility among components selected by the semen diluent is realized, and the problems that the sperm activity, deformity rate, storage time and cost comprehensive performance cannot be simultaneously combined are solved.

Drawings

FIG. 1 is a graph comparing sperm motility of different antibiotic diluent formulations;

FIG. 2 is a graph comparing sperm cell rates for different antibiotic semen dilutions;

FIG. 3 is a graph comparing antibiotic costs for each antibiotic formulation;

FIG. 4 is a schematic illustration of the effect of 5 semen dilutions on boar sperm motility at ambient temperature;

FIG. 5 is a schematic diagram showing the effect of 5 semen dilutions on the rate of boar sperm malformation at room temperature;

fig. 6 is a schematic diagram showing the effect of 5 semen dilutions on the linear motion of boar sperm stored at normal temperature.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The technical scheme of the scheme is further described through the specific embodiments with reference to the accompanying drawings.

1. Preparation of experiments

1-1, test reagent

Glucose, fructose, trehalose, lactose, citric acid, sodium citrate, sodium bicarbonate, ethylenediamine tetraacetic acid (EDTA-2 Na), tris, potassium chloride, taurine, glutathione (reducing), cysteine, L-glutamine, vitamin C, bovine Serum Albumin (BSA), beta-cyclodextrin, gentamicin, penicillin, streptomycin, compound neonomine, gentamicin sulfate, enrofloxacin.

1-2, laboratory apparatus

Electronic balance, 5000. Mu.L specification pipette, 1000. Mu.L specification pipette, 10. Mu.L specification pipette, 5ml centrifuge tube, 80ml insemination bottle, magapor sperm detection system, magapor detection plate, lei Ci pH meter PHSJ-3F model, optical microscope, and constant temperature water bath.

1-3 semen Source

The test semen is from Duroc, dabai, changbai of a boar station in Guangdong province, 2 heads each.

1-4, preparation before semen collection

1-4-1 preparation of tools

The prepared semen collection cup, latex glove, disposable PE glove, scissors, paper extraction (without fragrance and scraps), towel, bottle washing filled with normal saline, bucket, clear water, semen collection recording list, heat preservation foam box and electric kettle.

1-4-2, sperm collecting cup preparation

The semen collecting cup is required to be preheated in advance, filter paper is arranged, and the semen collecting cup is clean and pollution-free, and the semen collecting column is cleaned in a sanitary manner before semen collection; checking whether the fake sow table is stable or not, and carefully wiping the table surface of the fake sow table and the lower part of the rear body; the height of the fake sow table is adjusted according to the size of the boar; ensure that the rubber anti-skid pad is placed behind the fake sow table so as to ensure comfortable standing of the boar when the boar climbs across the fake sow table.

1-4-3, preparation of boar

Drilling a mining precision fence gate, and setting a channel; checking the boar column number and the ear number; checking the health condition of the boar, and judging whether the boar has lameness or fever; and opening a fence door, driving the boar by using a boar baffle plate, and prohibiting the boar from standing in front of the boar to drive the boar into the semen collection fence.

1-5 semen collection

Collecting semen of the boars;

1-6, extracting 1ml of semen diluted by each formula every day into a centrifuge tube, heating the centrifuge tube at 37 ℃ for 15-30 minutes in a constant-temperature water bath, and detecting the sperm motility and the deformity rate. The test was continued for 13 days.

1-6-1, sperm motility: the test adopts a sperm detection system of a Magapor company to carry out detection statistics on sperm which carry out linear motion on semen. The detection method refers to the experimental operation steps provided by the Magapor sperm detection system. The semen for test is firstly subjected to repeated up-down 5 times of shaking, then 1mL of each group of test semen is sucked by a pipetting gun and is filled into a 1.5mL centrifuge tube, marking is carried out, and the mixture is put into a water bath pot and heated for 15 minutes at 37 ℃. The semen in the 3.5. Mu.L centrifuge tube was then pipetted into a Magapor detection plate and then detected using a Magapor sperm detection system.

1-6-2, sperm malformation rate: the boar sperm malformation type mainly comprises the following types, namely distal end mass drop, proximal end mass drop and middle end tail folding. The CASA system of Magapor recognizes abnormal sperm when performing primordial detection, thereby obtaining the sperm abnormal rate. For each sample, 3 different fields were used for the vitality analysis, and 5 different fields were used for the deformity analysis.

1-6-3, data processing: one-way ANOVA analysis of variance was performed using GraphPad prism8.0 software and multiple comparisons were performed with P < 0.05 as a criterion for significance of the differences.

2. Comparative example a:

2-1, preparation of dilution

Weighing various components according to the formula dosage by using an electronic balance, adding the weighed reagent into a sterilized 1L beaker, weighing 1L ultrapure water, pouring into the beaker, fully dissolving the ultrapure water by using a magnetic stirrer, and then adding citric acid to adjust the pH value of the diluent to be between 6.4 and 6.5. After the dissolution is completed, pouring the mixture into a clean container, marking the mixture, sealing the mixture, and storing the mixture in a refrigerator at 4 ℃. The formulation is shown in Table 1;

table 1-dilution powder formulation of comparative example A1

2-2 dilution and preservation of semen

5 formula dilutions were added 5mL each to an 80mL semen bottle and marked, and placed in a 36.5 ℃ water bath for preheating. The clean semen bag is sleeved into a clean 1L beaker, and the freshly collected semen is taken out from the pneumatic conveying device, wherein the temperature of the semen is generally about 36.5 ℃.5mL semen is taken by a 5000-mu L specification pipetting gun and respectively added into marked semen bottles, the dilution is completed in a ratio of 1:1, and when the temperature of the semen in the semen bottles is equal to that of the diluent, the diluent of each formula is slowly poured into the semen bottles, and the volume is fixed to 80mL scale marks. And then slowly cooling the diluted semen in a room, transferring the diluted semen to a 17 ℃ refrigeration house after cooling to the room temperature, covering the cooled semen by using a towel, gently shaking a semen bottle once every 12 hours, detecting the semen every day, and recording the semen index.

2-3, results and analysis

2-3-1, effect of Diluent of different formulations on the viability of pig sperm preserved at Normal temperature

As can be seen from fig. 4, sperm motility was not significantly different in sperm diluted with five self-contained dilutions although the sperm motility was changed before and after dilution. Under the condition of preserving at 17 ℃, the energy of the third day after preserving reaches more than 0.6, and the comparative example A1, the comparative example A3 and the comparative example A4 are provided. The viability of comparative example A2 was already below 0.5 after the first day of storage, the effect of storage was significantly lower than that of comparative example A1 and comparative example A4 (P < 0.05), and there was no significant difference (P > 0.05) from comparative example A3 and comparative example A5. Comparative examples A1 and A4 showed sperm motility of 0.6 or higher on day 7 of storage. The preservation effect sequence of each formula diluent under the normal temperature preservation condition is as follows: comparative example A2-comparative example A3-comparative example A5-comparative example A1-comparative example A4.

2-3-2, effect of different formula diluents on normal temperature preservation sperm malformation rate

FIG. 5 is the effect of 5 semen dilutions on the rate of sperm malformation in normal temperature storage boars.

2-3-3, effect of Diluent of different formulations on Linear sperm movement

As can be seen from FIG. 6, the difference in elevation of sperm linear motion was not significant (P > 0.05) for each formulation before and after dilution. On the first to third days of storage, comparative example A3 was significantly different from comparative example A4, comparative example A5 (P > 0.05). On days five to eight of storage, there was a significant difference between comparative example A1 and comparative example A2, and comparative example A3 was a significant difference between comparative example A2, comparative example A4, and comparative example A5, but after 14 days of analysis, there was no significant difference in the effect of the five formulas on sperm linear motion (P > 0.05). The results show that the five formula dilutions have little effect on the linear motion of sperm.

2-4, discussion

2-4-1, boar semen dilution powder additive component effect

Many factors in the environment and operation can influence the preservation effect of semen and influence the preservation quality of sperms. At present, the pig semen cryopreservation technology is not mature, and the liquid normal temperature preservation technology is still a technology which needs to be emphasized at present. On one hand, the volume of the boar semen can be increased, the semen use times can be increased, the semen utilization rate of the excellent boar can be increased, and the cost can be saved; on the other hand, the method can provide nutrients, antioxidants and other components for the sperms, provide a proper living environment for the sperms, is favorable for prolonging the survival time of the sperms, reduces the stress injury to the sperms in long-distance transportation, and improves the fertilization capacity of the sperms. During preservation of porcine semen, the diluent components, the concentration of each component and the proportion of the components have important influence on the semen preservation effect.

Five formulas with different characteristics are designed in the test. Comparative example A1 contains energy substances such as glucose, fructose, and lactose as main components. The boar sperm can utilize saccharides in the semen to provide energy for self-movement, when the energy substances in the semen are consumed, the sperm begins to consume ATP stored in the online granulocyte, and the ATP content in the sperm cells is extremely small, so that the sperm can be completely consumed within 24 hours, and the activity rate of the sperm is reduced. The pig normal temperature preservation diluent is added with a proper amount of energy substances, so that the activity of sperms can be improved, and the preservation time of the sperms can be prolonged. Glucose is used as a common nutrient component of the diluent, the osmotic pressure of the diluent can be regulated, so that the damage to sperms caused by hypertonic or hypotonic environments is prevented, but the concentration of sugar is not too high, otherwise, the acrosome of sperms is damaged, and the fertilization effect is low.

Comparative example A2 mainly uses antioxidants such as glutathione and cysteine to counteract ROS produced by sperm respiration metabolism, and also adds a small amount of taurine, which acts to increase sperm viability and antioxidant capacity. However, the results show that the comparative example A2 has the worst preservation effect, the lowest preservation time and the highest variation of the deformity rate, and the reason for the results is that two or more antioxidants are possibly added together, so that the reduction effect in the diluent is too strong, thereby affecting the normal metabolism of sperms, damaging the plasma membrane of the sperms and being unfavorable for the normal survival of the sperms. The compatibility effect between different sperm protecting agents and antioxidants should be further studied, so that the normal-temperature preservation effect of boar semen is improved.

The comparative example A3 is prepared by adding BSA, vitamin C and L-glutamine, and the formula is mainly characterized by protecting sperm plasma membrane and improving sperm integrity. Among them, studies have shown that BSA has the effect of stabilizing osmotic pressure of diluent and protecting sperm cell membrane. Studies show that vitamin C is slightly acidic, so that the sperm can reduce the activity metabolism, and the effect of effectively preventing peroxidation of sperm phospholipids is achieved. It has also been found that the addition of vitamin C does not promote both the preservation of porcine semen at low temperature and at low temperature, but rather has an adverse effect on sperm when the vitamin C concentration is greater than 25. Mu.g/mL. It has also been found that vitamin C increases the time for preservation of sperm by reducing the plasma membrane fragility and permeability of sperm and inhibiting sperm motility. Also, researches show that vitamin C is an antioxidant substance and can react with ROS directly, so that the content of ROS is reduced, the adverse effect of ROS on sperms is reduced, the activity and fertility of sperms are improved effectively, and the structural integrity of sperms is improved. Thus, the addition of vitamin C to a diluent is currently not under the same theories and is under further investigation.

The comparative example A3 is also added with a small amount of beta-cyclodextrin, and the beta-cyclodextrin has a certain adsorption effect, can adsorb harmful products of sperm metabolism, prolongs the survival time of sperm, and simultaneously has the effect of protecting the plasma membrane of the sperm, and reduces the damage of the plasma membrane caused by oxidization.

Comparative example A4 is a combination of gentamicin and neonomine with better efficacy by replacing penicillin and streptomycin antibiotics. Penicillin is used as beta lactam antibiotics, has short action time and good antibacterial effect on gram-positive bacteria, but has no good antibacterial effect on escherichia coli, and streptomycin has antibacterial effect on gram-positive bacteria and is ineffective on escherichia coli. The research result shows that the compound neonomine is an antibiotic medicine with wider antibacterial spectrum and more remarkable antibacterial effect than penicillin, and has higher sensibility to enterobacteriaceae from different sources, and the optimal addition amount is 2.0g/L.

Comparative example A5 combines the characteristics of the 4 formulations above. However, the results showed that the effect of preserving sperm was not as good as that of comparative examples A1 and A4. The semen dilution powder component is further required to be studied.

2-4-2, study on the pH of the Diluent

At present, the pH value of the dilution liquid for in-vitro preservation of boar sperms is not unified, and researches are considered to be that the optimal pH value of the boar sperms preserved in vitro is 6.4, and the preservation effect of the semen pH value is considered to be better between 6.5 and 6.8. Therefore, the metabolism rate of sperm is reduced in weak acid environment, and the preservation effect is improved. However, it is considered that the in vitro preservation pH value of semen should have better preservation effect in a weak alkaline environment of about 7.0, because semen can be metabolized by glycolysis path, such as acid metabolite of lactic acid, while weak alkaline substances in the diluent can counteract the acid metabolite, prevent semen pH environment from changing into acid environment rapidly, cause acidosis of semen, influence normal function of semen, change structure of semen, and damage plasma membrane and acrosome of semen. In addition, the pH of the original semen is alkalescent, and the addition of alkalescent diluent can be in line with the original living environment of the sperms, so that the stimulation to the sperms is reduced. The pH value is in the process of preserving semen, the tendency that the area is stable after the semen is firstly reduced and then risen is presented, the semen possibly does not enter a dormant state in a period of time after the semen is diluted, the movement capacity is strong, the metabolism is vigorous, a large amount of acidic metabolites are produced to reduce the pH value of the semen, the semen is weak acid at the moment, the semen reduces the metabolism rate in the weak acid environment, energy substances in the diluent are also reduced, the buffer substances start to act, the pH value of the semen is not reduced any more, and finally the pH value of the semen reaches balance. Therefore, further studies are required as to whether the environment in which sperm is stored in vitro is weakly acidic or weakly basic, which environment has a good storage effect, and to select a suitable buffer substance.

2-5 conclusion

The results show that the preservation effect of the formula I is best by comprehensive sperm index analysis, and the formula I has the component characteristics that glucose, fructose and lactose are added, and non-reducing disaccharide trehalose with stable properties is also added, and the formula IV is the worst formula II. Based on the study of this comparative example, the formulation of example a was designed.

3. Example a:

weighing various components according to the formula dosage by using an electronic balance, adding the weighed reagent into a sterilized 1L beaker, weighing 1L ultrapure water, pouring into the beaker, fully dissolving the ultrapure water by using a magnetic stirrer, and then adding citric acid to adjust the pH value of the diluent to be between 6.4 and 6.5. After the dissolution is completed, pouring the mixture into a clean container, marking the mixture, sealing the mixture, and storing the mixture in a refrigerator at 4 ℃. The formulation is shown in Table 2;

TABLE 2 dilution formulation of example A

3. Results

3-1, comparison of semen viability preservation aging of different diluted powder formulas:

as is clear from FIG. 1, the number of days of storage for which the motility of comparative example A6 was 0.6 or more was 6 days, and the sperm motility was 0.6 or more for examples A1 to 10 days, and examples A2 and A3 were maintained until 11 days. Sperm motility of semen diluted by the four formula diluents is changed before and after dilution, but the difference is not obvious. Under the condition of preserving at 17 ℃, the activity of all the formulas can reach more than 0.6 in the sixth day after the semen is diluted. The preservation effect sequence of each formula diluent under the normal temperature preservation condition is as follows: example a2≡example A3> example A1> comparative example A6.

3-2, aging comparison of semen deformity rate of different diluted powder formulas:

with the increase of the preservation time, the aging effect of the four formula dilutions on the sperm malformation rate of pigs is as follows: as can be seen from FIG. 2, the sperm cell malformation rate of each formulation was maintained below 18% for at least 7 days, while comparative example A6, comparative example A5, was above 20% starting on day 8, example A1 was above 20% on day 13, example A3 was above 18% on day 13, and example A2 was still below 18% on day 13. With the increase of the preservation time, the rate of sperm malformation tends to increase. The sperm malformation rate in the four antibiotic dilutions was not significantly different 7 days ago. After 7 days, comparative example A6 began to be very significantly worse than examples A1, A2 and A3 (P < 0.01). On day 13, example A2 and example A3 were significantly better than example A1 (P < 0.05). The result shows that in the aspect of the deformity rate, the preservation effect of the diluent with different formulas is respectively as follows from high to low: example a2≡example A3> example A1> comparative example A6.

3-3, comparison of cost of different diluent powder formulas:

since all the formulation base dilutions were identical, only the antibiotic ingredients were different, the cost differences in antibiotic ingredients represented the cost differences for the diluted powder formulations, calculated as shown in table 3:

TABLE 3 antibacterial use costs of various antibiotic formulations

As can be seen, the enrofloxacin-containing example A3 is the least costly one, and has an antibiotic cost of about 0.3 yuan/L, whereas the gentamicin sulfate formulation of the most commonly used example A1 currently on the market has an antibiotic cost of about 0.74 yuan/L, which is nearly 60% more than that of the enrofloxacin formulation, and therefore, the enrofloxacin antibiotic formulation is a diluent powder formulation with the least antibiotic cost.

3-4, discussion:

3-4-1, action of antibiotics on semen preservation

Firstly, the influence analysis of the sperm motility and the sperm malformation rate by the sperm dilutions of 4 different formulas can be seen: the semen diluent formulation with the addition of antibiotics did extend the time of semen preservation, wherein gentamicin sulfate semen diluent formulation versus comparative example A5, the number of days of preservation with viability greater than 0.6 was extended by 4 days, while enrofloxacin semen diluent formulation and the green streptomycin semen diluent formulation versus comparative example A5, the time of preservation with viability greater than 0.6 was extended to 11 days.

Next, the sperm malformation rate of comparative example A5 was lower than 18% for 7 days, while the group to which gentamicin sulfate was added was prolonged to 12 days in comparison with comparative example A5. The group to which the green streptomycin was added and the group to which the enrofloxacin was added were prolonged to 13 days as compared with comparative example A5.

From the results of comparative example A5, it can be seen that: the abnormal rate of the semen is increased when the sperm motility is suddenly reduced, and the main reason is that the semen starts to be greatly reproduced on the sixth day, so that nutrition substances in the semen are contended, meanwhile, the semen steady state is damaged by various bacterial reproduction metabolic processes, including pH value, osmotic pressure and the like, so that the metabolism disorder of the sperm and the structure of the sperm are damaged, and the abnormal rate also starts to be obviously increased on the following day when the sperm motility is suddenly reduced. The reason why the vitality and the deformity rate of other formulas are relatively stable is that the antibiotics have sterilization or bacteriostasis effect on semen, during the semen preservation process, bacteria are destroyed at first or are partially survived when the semen is diluted, but the bacteria cannot realize growth and reproduction and are restrained within a certain range, so that nutrient substances and environmental homeostasis required for maintaining the sperms are preserved, and the sperms can be quickly revived and relatively stable sperm structures are maintained after the sperms are warmed. Thus the diluent of this example A was compared to comparative examples A1-A6, which significantly reduced sperm cell deformity. As apparent from the experimental results, the enrofloxacin and penicillin and streptomycin mixture has better preservation effect on semen diluent. This is closely related to the principle of its antimicrobial action, which is the inhibition of DNA deoxyribonucleic acid (DNA) gyrase by enrofloxacin, thereby affecting DNA replication and causing its death. Since bacterial cells belong to prokaryotic cells, DNA is naked, and livestock and poultry cells belong to eukaryotic cells, DNA is coated, so that the bacterial cells easily enter thalli and contact with the DNA, and selective reaction occurs.

The mechanism of action of penicillin is achieved by inhibiting the synthesis of cell wall mucins by bacteria. The sensitive bacteria in the growing period are most active in division, the cell wall is undergoing biosynthesis, and under the action of penicillin, the synthesis of mucin can not form the cell wall because of being blocked, and finally, the cell wall is cracked and dead under the action of osmotic pressure. In contrast, in bacteria in a non-growing stage, penicillin does not inhibit or kill the bacteria in this stage because there is no process of forming cell walls. The main antibacterial mechanism of streptomycin combined use with penicillin is to inhibit protein synthesis of bacteria, so that permeability of bacterial cell membranes is enhanced, and intracellular substances are extravasated to cause death of bacteria, and part of drug-resistant bacteria can cause death or inhibition of bacteria under the action of penicillin because penicillin is contained in diluent. The combined use of penicillin and streptomycin can achieve the effect of enhancing the antibacterial effect.

In addition, gentamicin belongs to the family of aminoglycosides, and besides spectinomycin, is the only currently known ribosomal inhibitor that causes protein misinterpretation. It inhibits the function of bacterial ribosomes, leading to bacterial death. It belongs to the same class of antibiotics as streptomycin, but has a broader antimicrobial spectrum than streptomycin. In this experiment, the activity of the formulation was maintained for more than 0.6 days less than that of the formulation of the green streptomycin, and the deformity rate was maintained for less than 18% for the same less than one day. It is presumed that the antibacterial spectrum of penicillin and streptomycin after combined application is larger than that of gentamicin, so that the antibacterial spectrum has inhibition and killing effects on more bacteria in semen.

As is evident from the experiment, the effect of the antibiotic on killing or inhibiting bacteria is greatly helpful for preserving semen, and the preservation effect of any antibiotic in the semen diluent formula is better than that of the semen diluent without the antibiotic in the comparative example A, and the activity and the deformity rate are more excellent than those of the semen diluent without the antibiotic in the comparative example A.

Cost analysis of 3-4-2, antibiotics

From the results of this experiment, it can be seen that the cost of the semen diluent added with antibiotics is between 0.3 yuan per liter and 1 yuan per liter, which is based on the unit price per liter of small production; however, if the method is used for mass production or can be taken to a lower wholesale price, the price does not deviate too much, and after all, the production cost of the antibiotics is different from the production process of each antibiotic, so that the cost result of the experiment has a certain reference significance for mass production of factories.

Wherein, the antibacterial cost of the enrofloxacin formulation is cheaper than that of other two antibiotic formulations, 50 g/bag of diluent powder is generally used in the market, each bag can be diluted into 1L of diluent, and only 0.1g of enrofloxacin is added into 1L of diluent, thus the cost is only 0.3 yuan. Compared with the 1-element cost of other green streptomycin, the 0.7-element cost of gentamicin sulfate has great cost advantage, and the semen preservation effect is equivalent to that of the green streptomycin, and can be preserved for 11 days. Enrofloxacin is therefore the first antimicrobial material for mass production of diluent powders.

3-4-3, conclusion

Among the formulas of antibiotic semen diluents such as gentamicin, green streptomycin and enrofloxacin, the semen diluent of the enrofloxacin formula has the best preservation effect on sperms, the vitality after dilution is more than 0.6 and can be maintained for 11 days, the deformity rate is less than 18% and can be maintained for 11 days, and the antibacterial cost is only 0.3 yuan per liter, so that the semen diluent is the optimal choice in the test.

The technical principle of the present solution is described above in connection with the specific embodiments. The description is only intended to explain the principles of the present solution and should not be construed in any way as limiting the scope of the present solution. Based on the explanations herein, other embodiments of the present solution will be apparent to those skilled in the art without undue burden, and such modifications will fall within the scope of the present solution.

Claims

1. The boar semen diluent is characterized by comprising the following components in parts by mass: 25-35 parts of glucose, 15-25 parts of fructose, 3-7 parts of lactose, 1-2 parts of trehalose, 1-2 parts of sodium bicarbonate, 5-7 parts of sodium citrate, 5-7 parts of Tris, 2-3 parts of EDTA-2Na, 0.1-1 part of potassium chloride and an antibiotic composition;

2. A method for preparing a boar semen diluent according to claim 1, comprising the steps of:

3. Use of a diluent in the preparation of a boar semen diluent having a low antimicrobial cost, wherein the diluent is a boar semen diluent according to claim 1.

4. Use of a diluent in the preparation of a boar semen diluent with a high preservation effect, characterized in that the diluent is a boar semen diluent according to claim 1.