CN101856016A - Diluent for preserving porcine semen at normal temperature - Google Patents
Diluent for preserving porcine semen at normal temperature Download PDFInfo
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Abstract
The invention relates to a diluent for preserving porcine semen at normal temperature. A total solute of the diluent comprises 45 to 55 percent by weight of monose combination, 15 to 25 percent by weight of amino acid combination and 25 to 35 percent by weight of electrolyte, buffer, antioxidant, stabilizing agent and antibiotics. Various substances are dissolved in double distilled water and a volume of 1, 000 milliliters is fixed so as to finally make an osmotic pressure of the diluent in the range of 330 to 360mosmo/L and make a pH value of the diluent in the range of 6.3 to 6.5. After the porcine semen is preserved at normal temperature in the environment of the diluent of the invention for 5 to 7 days, the viability can still be kept over 0.6 and the acrosom entire rate can reach 85 percent. When the porcine semen preserved by adopting the diluent of the invention is used for mating on 260 sows, the conception rate and the litter size reach 90.2 percent and 10.7 respectively and reach or exceed the levels of the import diluent. The diluent of the invention replaces the import bovine serum albumin with the monose combination and the amino acid combination, but the cost of the diluent of the invention is only 1/3 of that of the import diluent, so the production cost of artificial insemination of swines is reduced greatly.
Description
Technical field
The porcine semen at normal temperature that the present invention relates to a kind of artificial preparation is preserved compound dilution, is mainly used in the middle of the production breeding of pig industry.
Background technology
China began just semen at normal temperature preservation and the technology of artificial insemination of pig to be furtherd investigate and studied from the fifties, and through constantly improving and development over half a century, this technology has been tending towards maturation.At present, the seminal fluid of pig is preserved normal temperature preservation and low temperature preservation dual mode.Lower when low temperature is preserved owing to pig plasmalemmae of sperms phosphatid ylcholine content, and phosphatidyl-ethanolamine and sphingomyelin content are higher, it is asymmetric that cholesterol distributes, more in the plasma membrane outside, and cholesterol/phosphatide ratio is significantly low and other mammalian sperm.The sperm that these special physiological structures make pig is responsive especially to low temperature, and freezing preservation technical difficulty is big, and cost height, conception rate are lower, still under test so far.Therefore, the widespread usage semen at normal temperature is preserved technology in the production breeding of pig.The key of this technology is to adopt good dilution prescription, for sperm provides the abundant nutrition material, keeps constant osmotic pressure, pH value etc. is good living environment of creation of spermatid.Now, some developed countries such as the U.S., Italy, New Zealand, Japan etc. the seminal fluid of pig preserve and technology of artificial insemination aspect research and development comparatively ripe, developed short, in, long-acting sperm dilution liquid product.The short seminal fluid that can make was preserved 3～5 days, and middle effect can make seminal fluid preserve 5～7 days, long-actingly seminal fluid was preserved 7～10 days and big change does not take place vigor, and can keep good insemination rate and litter size.Technology of artificial insemination these national pigs has obtained popularizing comprehensively.In recent years, many researchers of China were also preserved aspects such as dilution prescription and insemination effect and had been carried out big quantity research to the normal temperature of pig seminal fluid, and had obtained bigger progress.But, up to the present, the material of keeping spermatid nutrition in the diluting boar semen formula of liquid of domestic development is all based on sugar and bovine serum albumin(BSA), cost is higher, and the dilution prescription that normal temperature is preserved the effect that still can keep in 3～5 days well inseminating is not obtained gratifying achievement (progress Fujian animal and veterinary second phase 9-11 in 2007 that Lin Yongli Xue Yong post boar semen is preserved as yet; Scarlet, Zheng Hong training porcine semen at normal temperature is preserved progress Heilungkiang animal reproduction first phase 16-19 in 2004; 2004 the 6th phase 46-49 of progress China's herding magazine that the pig sperm liquid-state such as Yue Kuizhong that soars is preserved; Open progress pig industry 2009 the 08th phases of science of pig normal temperature seminal fluid preservations such as the precious Ge Xinjun that sacrifices one's life for).Semen diluent of pig and artificial insemination product mostly are external import on China market.Costing an arm and a leg of import dilution, general pig farm is difficult to bear.And the dilution quality that oneself is prepared can't guarantee that the dilution of import is still adopted on each big pig farm.This will increase the test-tube production cost of China's pig industry greatly, influence the popularization of the technology of artificial insemination of pig, and obviously, the seminal fluid of pig is preserved popularizing of swine artificial insemination technology very important.At present, along with the fast development of pig industry, can predict, the technology of artificial insemination of China pig will overall application and universal in the coming years.Development has the semen diluent import substitutes of China's independent intellectual property right super quality and competitive price, reduces the artificial insemination production cost of pig, to promoting China's pig industry healthy and sustainable development significant.
Summary of the invention
The present invention plays and supplies with the nutrition of pig spermatid; keep protective effects such as its osmotic pressure, pH value; thereby enlarge pig seminal fluid capacity, prolong the pig seminal fluid holding time, porcine semen at normal temperature is preserved still can be kept better vigor and acrosomal integrity in 5～7 days, and keep good insemination rate and litter size.
Technical scheme of the present invention is: a kind of diluent for preserving porcine semen at normal temperature, the main component of supplying with pig spermatid nutriment is in the solute of this dilution: monose combination and amino acid combination, the weight of monose combination is 45%～55% of total soluble matters weight, and the weight of amino acid combination is 15%～25% of total soluble matters weight; All the other solutes of 25%～35% are electrolyte, cushion and antioxidant, stabilizing agent, antibiotic; Described monose combination, amino acid combination, electrolyte, cushion and antioxidant, stabilizing agent, antibiotic is dissolved in the distilled water and be settled to 1000 milliliters, finally makes the osmotic pressure of described dilution remain on 330～360mosmo/L, pH maintains 6.3～6.5.
Described monose combination is: glucose, sucrose, fructose, lactose, trehalose; Described amino acid combination is: glycine, glutamic acid, arginine, a word used in person's names propylhomoserin; Described electrolyte, cushion comprise citric acid, sodium citrate, sodium bicarbonate, sodium dihydrogen phosphate, tri methylol amino methane, potassium chloride, magnesium chloride; Described antioxidant comprises vitamin E, cysteine; Described stabilizing agent and antibiotic comprise sodium ethylene diamine tetracetate, penicillin, streptomycin.
The preferred solute prescription and the weight portion thereof of described dilution are: glucose 3～8g, sucrose 1～5g, fructose 1～4g, lactose 10～15g, trehalose 1～3g, glycine 1～4g, glutamic acid 3～5g, arginine 1～3g, a word used in person's names propylhomoserin 0.5～1.5g, citric acid 1～3g, sodium citrate 3～5g, sodium bicarbonate 2～4g, sodium dihydrogen phosphate 1～3g, tri methylol amino methane 1～5g, potassium chloride 0.2～0.5g, magnesium chloride 0.3～0.5g, vitamin E 0.3～0.5g, cysteine 0.2～0.5g, sodium ethylene diamine tetracetate 1～2g, penicillin 0.07～0.10g, streptomycin 0.05～0.07g; Be settled to 1000mL with distilled water at last.
The present invention is mainly used in the middle of the production breeding of pig industry, supply with spermatid nutrition by playing, keep protective effects such as its osmotic pressure, pH value, reach expansion pig seminal fluid capacity, prolong the pig seminal fluid holding time, increase sow and the conception rate and the nest litter size of joining a number, raising sow; Porcine semen at normal temperature is preserved still can be kept better vigor and acrosomal integrity in 5～7 days, and keep good insemination rate and litter size.
The present invention can improve to greatest extent the good species boar availability, reduce production costs, bring into play the pig industry maximum economic benefits.Particularly accelerate and promote aspect the application of technology of artificial insemination of pig and the popularization significant.
The advantage of maximum of the present invention be with monose combination and amino acid combination replacement the bovine serum albumin(BSA) in the import dilution, its cost has only 1/3 of import dilution, reduced the artificial insemination production cost of pig, to promoting China's pig industry healthy and sustainable development significant.
The present invention is as follows the test data in 2009～2010 years: the seminal fluid of pig normal temperature in dilution environment of the present invention preserves that 5～7 days vigor still can remain on more than 0.6, the percentage of head rice of acrosome can reach 85%, sees Table 1; Adopt the present invention and join 260 of sows, conception rate and litter size reach 90.2% and 10.7 respectively, see Table 2.Conception rate and litter size meet or exceed import dilution level.
Table 1 dilution sperm viability of the present invention, acrosomal integrity
| Time | Vigor | Plasma membrane percentage of head rice % | Acrosomal integrity % |
| First day | ??0.8 | ??95 | ??93 |
| Second day | ??0.75 | ??93.4 | ??91 |
| The 3rd day | ??0.75 | ??92.5 | ??90.5 |
| The 4th day | ??0.7 | ??90.6 | ??89 |
| Time | Vigor | Plasma membrane percentage of head rice % | Acrosomal integrity % |
| The 5th day | ??0.65 | ??89.5 | ??87.6 |
| The 6th day | ??0.65 | ??88 | ??87 |
| The 7th day | ??0.65 | ??87.5 | ??86 |
Table 2 2009-2010 artificial insemination of the present invention result of the test
| A semen deposition number | A number of becoming pregnant | Conception rate (%) | The farrowing of mean nest | |
| 2009 | ??135 | ??119 | ??89.3 | ??10.5 |
| 2010 | ??167 | ??141 | ??91.1 | ??10.9 |
| Amount to | ??302 | ??260 | ??90.2 | ??10.7 |
Embodiment
It is pure that used chemical reagent is the high-quality analysis among the embodiment.
Embodiment 1, a kind of diluent for preserving porcine semen at normal temperature, its solute prescription and weight portion thereof are: glucose 5.5g, sucrose 1.5g, fructose 1.6g, lactose 15.0g, trehalose 1.5g, glycine 3.2g, glutamic acid 3.8g, arginine 1.5g, a word used in person's names propylhomoserin 1.0g, citric acid 1.8g, sodium citrate 3.3g, sodium bicarbonate 2.2g, sodium dihydrogen phosphate 1.2g, tri methylol amino methane 4.1g, potassium chloride 0.25g, magnesium chloride 0.32g, vitamin E 0.5g, cysteine 0.5g, sodium ethylene diamine tetracetate 1.5g, penicillin 0.07g, streptomycin 0.05g; Above-mentioned medicine is carried out accurate weighing, add distilled water and be settled to 1000mL, 36 ℃ of water-baths were dissolved 1 hour.
Embodiment 2, a kind of diluent for preserving porcine semen at normal temperature, its solute prescription and weight portion thereof are: glucose 3.8g, sucrose 5.0g, fructose 3.2g, lactose 10.0g, trehalose 2.5g, glycine 3.0g, glutamic acid 4.2g, arginase 12 .2g, a word used in person's names propylhomoserin 1.2g, citric acid 1.2g, sodium citrate 3.6g, sodium bicarbonate 3.7g, sodium dihydrogen phosphate 2.6g, tri methylol amino methane 1.7g, potassium chloride 0.35g, magnesium chloride 0.45g, vitamin E 0.35g, cysteine 0.45g, sodium ethylene diamine tetracetate 1.0g, penicillin 0.1g, streptomycin 0.07g; Above-mentioned medicine is carried out accurate weighing, add distilled water and be settled to 1000mL, 36 ℃ of water-baths were dissolved 1 hour.
Embodiment 3, a kind of diluent for preserving porcine semen at normal temperature, its solute prescription and weight portion thereof are: glucose 7.3g, sucrose 2.6g, fructose 1.2g, lactose 12.0g, trehalose 1.5g, glycine 3.5g, glutamic acid 3.8g, arginase 12 .2g, a word used in person's names propylhomoserin 0.5g, citric acid 2.2g, sodium citrate 4.1g, sodium bicarbonate 2.5g, sodium dihydrogen phosphate 1.5g, tri methylol amino methane 2.6g, potassium chloride 0.43g, magnesium chloride 0.5g, vitamin E 0.3g, cysteine 0.2g, sodium ethylene diamine tetracetate 1.2g, penicillin 0.08g, streptomycin 0.06g; Above-mentioned medicine is carried out accurate weighing, add distilled water and be settled to 1000mL, 36 ℃ of water-baths were dissolved 1 hour.
Preserve the process of pig seminal fluid with the present invention:
1. the collection of seminal fluid is manually held semen collection method collecting semen with detecting the false pig platform of employing, send the laboratory to carry out projects such as vigor, motility rate, density, color and luster smell after the semen collection immediately and detects.
2. the qualified seminal fluid of the dilution of seminal fluid detection dilutes immediately.Dilution according to sperm viability and density and above configuration carries out 1～10 times of dilution, and every milliliter contains sperm 10 alive
6Dilution process: dilution is joined in the seminal fluid along the slow gradation of glass bar.For the first time, carry out dilution second time at interval after 1 minute, add the dilution of twice for the second time, for the third time all addings with the dilution of seminal fluid equivalent.Gradation adds makes sperm can better adapt to the living environment of dilution.Seminal fluid mixes with dilution and must carry out under same temperature, and notes reducing the time that seminal fluid contacts with air as far as possible.
3. seminal fluid is preserved the dilution back and is used remaining seminal fluid to wrap up with 3-4 layer towelling coverlet to put into the preservation of slowly lowering the temperature of 15-18 ℃ of refrigerator.Shook once death between storage life in per 12 hours gently to reduce the long dormancy of sperm.Make sperm outstanding from shaking gently after refrigerator takes out during semen deposition once more, the packing semen deposition.
Claims (4)
1. diluent for preserving porcine semen at normal temperature, it is characterized in that: the main component of supplying with pig spermatid nutriment is in the solute of described dilution: monose combination and amino acid combination, the weight of monose combination is 45%～55% of total soluble matters weight, and the weight of amino acid combination is 15%～25% of total soluble matters weight; All the other solutes of 25%～35% are electrolyte, cushion and antioxidant, stabilizing agent, antibiotic; Described monose combination, amino acid combination, electrolyte, cushion and antioxidant, stabilizing agent, antibiotic is dissolved in the distilled water and be settled to 1000 milliliters, finally makes the osmotic pressure of described dilution remain on 330～360mosmo/L, pH maintains 6.3～6.5.
2. diluent for preserving porcine semen at normal temperature according to claim 1 is characterized in that: described monose combination is: glucose, sucrose, fructose, lactose, trehalose; Described amino acid combination is: glycine, glutamic acid, arginine, a word used in person's names propylhomoserin; Described electrolyte, cushion comprise citric acid, sodium citrate, sodium bicarbonate, sodium dihydrogen phosphate, tri methylol amino methane, potassium chloride, magnesium chloride; Described antioxidant comprises vitamin E, cysteine; Described stabilizing agent and antibiotic comprise sodium ethylene diamine tetracetate, penicillin, streptomycin.
3. diluent for preserving porcine semen at normal temperature according to claim 2 is characterized in that: the preferred solute prescription and the weight portion thereof of described dilution are: glucose 3～8g, sucrose 1～5g, fructose 1～4g, lactose 10～15g, trehalose 1～3g, glycine 1～4g, glutamic acid 3～5g, arginine 1～3g, a word used in person's names propylhomoserin 0.5～1.5g, citric acid 1～3g, sodium citrate 3～5g, sodium bicarbonate 2～4g, sodium dihydrogen phosphate 1～3g, tri methylol amino methane 1～5g, potassium chloride 0.2～0.5g, magnesium chloride 0.3～0.5g, vitamin E 0.3～0.5g, cysteine 0.2～0.5g, sodium ethylene diamine tetracetate 1～2g, penicillin 0.07～0.10g, streptomycin 0.05～0.07g; Be settled to 1000mL with distilled water at last.
4. diluent for preserving porcine semen at normal temperature according to claim 3 is characterized in that: the optimum solute prescription and the weight portion thereof of described dilution are: glucose 5.5g, sucrose 1.5g, fructose 1.6g, lactose 15.0g, trehalose 1.5g, glycine 3.2g, glutamic acid 3.8g, arginine 1.5g, a word used in person's names propylhomoserin 1.0g, citric acid 1.8g, sodium citrate 3.3g, sodium bicarbonate 2.2g, sodium dihydrogen phosphate 1.2g, tri methylol amino methane 4.1g, potassium chloride 0.25g, magnesium chloride 0.32g, vitamin E 0.5g, cysteine 0.5g, sodium ethylene diamine tetracetate 1.5g, penicillin 0.07g, streptomycin 0.05g; Be settled to 1000mL with distilled water at last.
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