CN105325400A - Application of taurine in preparation of diluent for porcine semen preservation - Google Patents
Application of taurine in preparation of diluent for porcine semen preservation Download PDFInfo
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- CN105325400A CN105325400A CN201510711930.2A CN201510711930A CN105325400A CN 105325400 A CN105325400 A CN 105325400A CN 201510711930 A CN201510711930 A CN 201510711930A CN 105325400 A CN105325400 A CN 105325400A
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Abstract
The invention relates to application of taurine in preparation of a diluent for porcine semen preservation and further relates to a diluent for porcine semen preservation. The diluent for porcine semen preservation is prepared from glucose, sodium bicarbonate, sodium citrate, EDTA, citric acid, potassium chloride, sulfamethoxazole, gentamicin and taurine. The diluent for porcine semen preservation has the good and reliable preservation effect, can provide high-quality normal-temperature preserved porcine semen for artificial swine insemination, the vitality of the porcine sperms preserved by adopting the diluent for porcine semen preservation at the temperature of 17 DEG C is above 0.6 after 5 days.
Description
Technical field
The invention belongs to biological technical field, the normal temperature relating to animal semen preserves dilution, particularly dilution of preserving of a kind of porcine semen at normal temperature and preparation method thereof.
Background technology
Along with the propagation and employment of technology of artificial insemination in Intensive Farm of Pig Raising of pig, the Techniques of preserving of seminal fluid more and more comes into one's own, and day by day demonstrates it in many-sided advantages such as the reduction boar number of animals raised and feeding cost, the breed improvement of quickening low yield swinery, minimizing transmissions.Compared with frozen semen, there is after the artificial insemination of normal temperature liquid storage seminal fluid the advantages such as sow breeding does well in (conception rate, parturition rate and nest litter size are high), required expense is low, operation sequence is simple; Consider from the preservation effect of seminal fluid, the seminal fluid of normal temperature liquid storage is more conducive to the breeding of pig, can produce larger economic benefit.Therefore, in current production practices, the artificial insemination of pig mainly adopts the seminal fluid that normal temperature is preserved.
Porcine semen at normal temperature preserve be by the Semen routine of collection under the room temperature with certain mobility scale, utilize the activity of the sour environment Inhibit sperm of certain limit, reduce the energy ezpenditure of himself, make sperm remain on reversible state and not lose its fertility.In semen at normal temperature is preserved; the preservation effect of seminal fluid is subject to the impact of the many factors such as storage temperature, pH, osmotic pressure, extension rate, active oxygen; semen diluent can not only provide energy needed for metabolism for sperm; adjustment pH and osmotic pressure; the breeding of microorganism in seminal fluid can also be suppressed, play the effect of protection sperm.Therefore, dilution is the key factor affecting porcine semen at normal temperature preservation effect, closely bound up with the effect of being impregnated of sow.
In addition, diluting boar semen preserves formula kind much both at home and abroad at present, due to antibiotic a large amount of use, causes most of bacterium in seminal fluid to produce drug resistance, show the shortcomings such as effect instability, high cost, bring a lot of inconvenience to production field and basic unit's breed improvement station and specialist.
Summary of the invention
For defect or the deficiency of prior art, the invention provides the application of taurine for the preparation of pig semen preservation dilution.
Further, taurine coordinates antibacterials for the preparation of the application of pig semen preservation dilution.
Optionally, antibacterials of the present invention are SMZco and gentamicin.
The present invention additionally provides a kind of pig semen preservation dilution simultaneously, and the pig semen preservation dilution provided comprises taurine, glucose and antibacterial substance.
The present invention additionally provides a kind of pig semen preservation dilution simultaneously, and the pig semen preservation dilution provided comprises taurine, glucose, buffer substance, chelating agent, SMZco, gentamicin and ionic equilibrium material.
Preferably, of the present invention provided pig semen preservation dilution comprises: glucose, sodium bicarbonate, sodium citrate, EDTA, citric acid, potassium chloride, SMZco, gentamicin and taurine.
Optionally, between the component that of the present invention provided pig semen preservation dilution comprises and component with magnitude relation be: containing following material in every 1L aqueous solution: glucose: 35.0 ~ 40.0g, sodium bicarbonate: 0.5 ~ 1.0g, sodium citrate: 6.0 ~ 7.0g, EDTA:1.0 ~ 1.5g, citric acid: 0.1 ~ 0.5g, potassium chloride: 0.5 ~ 1.0g, gentamicin: 2 × 10
5iU, SMZco: 2.5 × 10
4iU, taurine: 5mmol.
Optionally, the pH of of the present invention provided pig semen preservation dilution is 6.80 ± 0.05, osmotic pressure is 300 ± 10mOsm.
SMZco, gentamicin and taurine conbined usage have synergy, can extend the Semen routine time.Result of the test of the present invention shows; preserve in dilution powder at pig semen and add SMZco, gentamicin and taurine; preserve after 5 days; sperm motility rate, plasm membrane integrity, acrosomal integrity, T-AOC content are higher than the experimental group of single use SMZco, gentamicin and single use taurine; and MDA concentration is lower than the experimental group of single use SMZco, gentamicin and single use taurine, wherein sperm motility rate, acrosomal integrity, MDA concentration effect are remarkable.
Pig semen preservation dilution of the present invention, preservation effect is good and reliable, and high-quality high-quality normal temperature can be provided to preserve seminal fluid for swine artificial insemination, preserve at 17 DEG C, after 5 days, sperm viability is more than 0.6.
Accompanying drawing explanation
Fig. 1 is single Tau and coordinates the concentration of Tau on the impact of T-AOC in seminal fluid;
Fig. 2 is single Tau and coordinates the concentration of Tau on the impact of MDA concentration in seminal fluid.
The concrete test of giving below in conjunction with accompanying drawing and inventor and using method, the present invention is described in further detail.
Embodiment
The dilution preserved for porcine semen at normal temperature of the present invention, answering matching while using, can be prepare at least 2 hours before sperm dilution.
Below in an example, the dilution that porcine semen at normal temperature is preserved needs matching while using.
Embodiment:
(1) diluent preparing of porcine semen at normal temperature preservation
The dilution for porcine semen at normal temperature preservation that the present embodiment provides is the aqueous solution, containing following material in every 1L aqueous solution:
Glucose: 35.0 ~ 40.0g, sodium bicarbonate: 0.5 ~ 1.0g, sodium citrate: 6.0 ~ 7.0g, EDTA:1.0 ~ 1.5g, citric acid: 0.1 ~ 0.5g, potassium chloride: 0.5 ~ 1.0g, gentamicin: 2 × 10
5iU, SMZco: 2.5 × 10
4iU, Tau:5mmol.
Preparation method comprises the following steps:
(1) take glucose by formula, sodium bicarbonate, sodium citrate, EDTA, citric acid, potassium chloride, SMZco, gentamicin, Tau are for subsequent use;
(2) glucose, sodium bicarbonate, sodium citrate, EDTA, citric acid, potassium chloride 1L distilled water are mixed with the aqueous solution;
(3) after each composition in the aqueous solution fully dissolves, add the gentamicin of formula ratio, SMZco and taurine (Tau), obtain the dilution preserved for porcine semen at normal temperature.
In order to determine the suitableeest interpolation concentration of Tau in diluent for preserving porcine semen at normal temperature and the conbined usage effect with two kinds of antibacterials, this test is provided with following scheme:
1, control group 1.: single antibacterials pig semen preserves dilution (containing following material in dilution: glucose: 36.0g, sodium bicarbonate: 0.6g, sodium citrate: 6.2g, EDTA:1.2g, citric acid: 0.2g, potassium chloride: 0.6g, gentamicin: 2 × 10
5iU, SMZco: 2.5 × 10
4iU, not containing Tau).
2, control group 2.: single Tau pig semen preserves dilution (containing following material in dilution: glucose: 36.0g, sodium bicarbonate: 0.6g, sodium citrate: 6.2g, EDTA:1.2g, citric acid: 0.2g, potassium chloride: 0.6g, Tau: be respectively 0.5mmol/L, 1mmol/L, 5mmol/L and 10mmol/L).
3, experimental group: Tau coordinates antibacterials pig semen to preserve dilution (containing following material in dilution: glucose: 36.0g, sodium bicarbonate: 0.6g, sodium citrate: 6.2g, EDTA:1.2g, citric acid: 0.2g, potassium chloride: 0.6g, gentamicin: 2 × 10
5iU, SMZco: 2.5 × 10
4iU, Tau: be respectively 0.5mmol/L, 1mmol/L, 5mmol/L and 10mmol/L).
Accurately deserve to be called with electronic analytical balance and state components dissolved in 1L distilled water, even by magnetic stirrer, namely make the dilution that porcine semen at normal temperature is preserved, put into 37 DEG C of water-baths for subsequent use.
(2) collection of seminal fluid
Use hand grip semen collection, collect stage casing concentrating part, be stored in thermos cup after filtering and take back laboratory.Microscopy at 37.5 DEG C immediately, observes sperm motility rate, and carries out colorimetric with spectrophotometer at 550nm place, calculates sperm concentration.Select color and luster and smell normally, sperm motility rate is more than 0.8, and density is 3 × 10
8~ 5 × 10
8the seminal fluid of individual/mL is used for normal temperature and preserves.
(3) sperm dilution
The temperature consistent with seminal fluid temperature (both temperature difference are within 1 DEG C) of the dilution 1, regulating porcine semen at normal temperature to preserve.Attention must with the temperature of seminal fluid for standard, and the temperature of the dilution then regulating porcine semen at normal temperature to preserve, can not contrary operation.
2, the dilution that the porcine semen at normal temperature configured is preserved slowly is added in seminal fluid along chamber wall, can not contrary operation, stir evenly gently or shake up.
3, according to production requirement determination extension rate, as need dilution for many times be done, first carry out the dilution of 1:1 low power, again the dilution that the porcine semen at normal temperature of remainder is preserved slowly is added after 1 minute, and mix.
4, wrap up with four layers of towel after dilution, make its slow cooling, room temperature checks sperm motility rate after placing 2 hours, if sperm motility rate does not obviously reduce before and after diluting again, puts into 17 DEG C of constant temperature storage boxes preserve together with towel.
(4) normal temperature preserves pig semen flavor evaluation
From insulating box, took out the seminal fluid that normal temperature is preserved every 24 hours, at the temperature of 37 DEG C, evaluate semen quality, main testing index is as follows:
1, sperm motility rate.Drawing 10 μ L seminal fluid with liquid-transfering gun is placed on slide, immediately at 400 times of optical microphotograph Microscopic observations after covered.Each sample random observation 3 visuals field, the number of sperm detecting visual field cathetus forward travel accounts for the ratio of total number of sperm, asks its mean value as sperm motility rate (see table 1).
2, acrosomal integrity.Draw 30 μ L seminal fluid with liquid-transfering gun, smear, after natural seasoning, under room temperature, fix 10 minutes with methyl alcohol, on each semen smear, then drip Ji's nurse Sa dye liquor respectively, dye 2 hours at 37 DEG C.Use running water loose colour afterwards, by the acrosome situation (see table 2) of microexamination sperm after natural seasoning.
3, plasm membrane integrity.Draw 1mL seminal fluid with liquid-transfering gun, add 9mL sodium citrate-fructose hypotonic medium respectively, bathe 30 minutes at 37 DEG C of Water Unders, and then draw one with micropipettor, in 400 times of curved tail rates (see table 3) of optical microphotograph Microscopic observation sperm.
4, measure the TAC (T-AOC) in the 1st day to the 5th day dilution seminal fluid of Semen routine and malonaldehyde (MDA) content, assay method carries out (see Fig. 1 and 2) according to kit specification.
(5) evaluation result
The dilution utilizing porcine semen at normal temperature of the present invention to preserve and store method, the evaluation result of its preservation effect is as follows:
The single Tau of table 1 and coordinate different Tau concentration on the impact of Boar spermatozoa motility rate
Note: the identical lowercase alphabet differential of colleague's data shoulder mark is different, and significantly (P > 0.05) different lowercase alphabet does not show significant difference (P < 0.05), lower same.
The single Tau of table 2 and coordinate different Tau concentration on the impact (%) of Boar spermatozoa acrosomal integrity
The single Tau of table 3 and the different Tau concentration of cooperation are on the impact (%) of Boar spermatozoa plasm membrane integrity
As shown in table 1, compared with control group, experimental group Boar spermatozoa motility rate generally higher than contrast 1., 2. group, and the Tau adding 1mmol/L and 5mmol/L in basal liquid can significantly improve sperm motility rate, extend sperm effective holding time (P<0.05), wherein 5mmol/L better effects if.
As shown in table 2, compared with control group, experimental group Sperm acrasomal integvity generally higher than contrast 1., 2. group, and the Tau adding 1mmol/L and 5mmol/L in basal liquid can significantly improve Sperm acrasomal integvity, extend sperm effective holding time (P<0.05), wherein 5mmol/L better effects if.
As shown in table 3, not obvious to plasmalemmae of sperms effect when adding Tau and antibacterials in basal liquid, difference remarkable (P>0.05) between plasmalemmae of sperms percentage of head rice experimental group and control group generally, but experimental group Sperm acrasomal integvity is generally higher than control group, and effect is better when the addition of Tau is 5mmol/L.
As shown in Figure 1, compared with control group, experimental group can improve the concentration of T-AOC in porcine semen at normal temperature preservation, and presents the rear reduction trend of irregular first increase with the increase T-AOC concentration of Tau dosage; Wherein, when the addition of experimental group Tau is 5mmol/L, T-AOC concentration is the highest, but generally with other processed group differences not significantly (P>0.05).
As shown in Figure 2, with contrast 1. compared with group, 2. contrast is organized and the Tau of experimental group interpolation 0.5mmol/L, 1mmol/L and 5mmol/L all can make MDA in seminal fluid decline, the wherein 5mmol/L Be very effective (P<0.05) of experimental group, and the Tau adding other processed group dosage effectively can not change the trend (P>0.05) that MDA content increases.
Can be drawn by above result, the Tau adding 5mmol/L in dilution powder formula of the present invention coordinates 2 × 10
5the gentamicin of IU and 2.5 × 10
4sMZco energy significant prolongation Boar spermatozoa effective holding time of IU, improve T-AOC content in sperm motility rate, acrosomal integrity and seminal fluid, reduce the generation (P<0.05) of MDA, effect is better than single interpolation antibiotic and Tau, but on membrane integrity impact little (P>0.05), sperm effective holding time is 5 days.After 5 days preserve, sperm motility rate, acrosomal integrity and plasm membrane integrity be respectively 0.67,80.55% and the content of 57.31%, T-AOC and MDA be respectively 4.76U/mL and 1.63nmol/mL.Therefore, 5mmol/LTau, 2 × 10 is added
5iU gentamicin and 2.5 × 10
4this patent dilution powder preservation effect of IU SMZco is good and reliable, high-quality high-quality normal temperature can be provided to preserve seminal fluid for swine artificial insemination.
Claims (8)
1. taurine is for the preparation of the application of pig semen preservation dilution.
2. taurine coordinates antibacterials for the preparation of the application of pig semen preservation dilution.
3. apply as claimed in claim 2, it is characterized in that, described antibacterials are SMZco and gentamicin.
4. a pig semen preservation dilution, is characterized in that, described pig semen preservation dilution comprises taurine, SMZco, gentamicin and glucose.
5. a pig semen preservation dilution, is characterized in that, described pig semen preservation dilution comprises taurine, SMZco, gentamicin, glucose, buffer substance, chelating agent and ionic equilibrium material.
6. pig semen preservation dilution as claimed in claim 5, it is characterized in that, described pig semen preservation dilution comprises: glucose, sodium bicarbonate, sodium citrate, EDTA, citric acid, potassium chloride, SMZco, gentamicin and taurine.
7. pig semen preservation dilution as claimed in claim 6, it is characterized in that, between the component that described pig semen preservation dilution comprises and component with magnitude relation be: containing following material in every 1L aqueous solution: glucose: 35.0 ~ 40.0g, sodium bicarbonate: 0.5 ~ 1.0g, sodium citrate: 6.0 ~ 7.0g, EDTA:1.0 ~ 1.5g, citric acid: 0.1 ~ 0.5g, potassium chloride: 0.5 ~ 1.0g, gentamicin: 2 × 10
5iU, SMZco: 2.5 × 10
4iU, taurine: 5mmol.
8. pig semen preservation dilution as claimed in claim 7, is characterized in that, the pH of described pig semen preservation dilution is 6.80 ± 0.05, osmotic pressure is 300 ± 10mOsm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107751185A (en) * | 2017-10-19 | 2018-03-06 | 界首市起点家庭农场 | One boar high-quality semen diluent |
| CN115918642A (en) * | 2022-12-05 | 2023-04-07 | 李苑有 | Protective agent for preserving boar semen at 4-20 ℃, preparation and preservation method and application |
| CN117296830A (en) * | 2023-09-25 | 2023-12-29 | 仲恺农业工程学院 | Boar semen diluent and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1349797A (en) * | 2000-09-11 | 2002-05-22 | Imv技术公司 | Attenuant for preserving pig's sperm |
| CN103445882A (en) * | 2013-08-28 | 2013-12-18 | 武汉市畜牧兽医科学研究所 | Method for improving moveability of sperm in frozen bull semen and in vitro fertilization rate |
-
2015
- 2015-10-28 CN CN201510711930.2A patent/CN105325400A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1349797A (en) * | 2000-09-11 | 2002-05-22 | Imv技术公司 | Attenuant for preserving pig's sperm |
| CN103445882A (en) * | 2013-08-28 | 2013-12-18 | 武汉市畜牧兽医科学研究所 | Method for improving moveability of sperm in frozen bull semen and in vitro fertilization rate |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107751185A (en) * | 2017-10-19 | 2018-03-06 | 界首市起点家庭农场 | One boar high-quality semen diluent |
| CN115918642A (en) * | 2022-12-05 | 2023-04-07 | 李苑有 | Protective agent for preserving boar semen at 4-20 ℃, preparation and preservation method and application |
| CN117296830A (en) * | 2023-09-25 | 2023-12-29 | 仲恺农业工程学院 | Boar semen diluent and preparation method and application thereof |
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Application publication date: 20160217 |