AU2021102920A4 - Base diluent for porcine frozen semen and preparation method and application thereof - Google Patents
Base diluent for porcine frozen semen and preparation method and application thereof Download PDFInfo
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- 210000000582 semen Anatomy 0.000 title claims abstract description 205
- 239000003085 diluting agent Substances 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims description 22
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 42
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 38
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 19
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 15
- 229930182821 L-proline Natural products 0.000 claims abstract description 15
- 229960002429 proline Drugs 0.000 claims abstract description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 14
- 239000007983 Tris buffer Substances 0.000 claims abstract description 14
- 239000012154 double-distilled water Substances 0.000 claims abstract description 14
- 239000008103 glucose Substances 0.000 claims abstract description 14
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 12
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 12
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 10
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 10
- 235000013345 egg yolk Nutrition 0.000 claims abstract description 10
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 10
- 229930182555 Penicillin Natural products 0.000 claims abstract description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 6
- 235000011187 glycerol Nutrition 0.000 claims abstract description 6
- 229940049954 penicillin Drugs 0.000 claims abstract description 6
- 229960005322 streptomycin Drugs 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 4
- 229960005150 glycerol Drugs 0.000 claims abstract 2
- 230000008014 freezing Effects 0.000 claims description 36
- 238000007710 freezing Methods 0.000 claims description 35
- 230000035899 viability Effects 0.000 claims description 30
- 238000004321 preservation Methods 0.000 claims description 25
- 239000000203 mixture Substances 0.000 claims description 24
- 239000002577 cryoprotective agent Substances 0.000 claims description 20
- 239000000463 material Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 13
- 210000004185 liver Anatomy 0.000 claims description 13
- 235000015277 pork Nutrition 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 11
- 235000020183 skimmed milk Nutrition 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000005057 refrigeration Methods 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
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- 239000007798 antifreeze agent Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
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- 239000006059 cover glass Substances 0.000 description 1
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- 238000001739 density measurement Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- 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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a base diluent for porcine frozen semen, comprising glucose, citric acid,
cholesterol, L-proline, Tris and double distilled water, further comprising egg yolk, penicillin, streptomycin,
glycerin and Equex.STM.
Description
Description
Technical Field
The present invention belongs to the technical field of agricultural biological breeding, and particularly relates to a diluent formula of porcine frozen semen.
Background
Over the last few decades, due to introduction and large-scale feeding of massive foreign pig breeds, the number of local pig breeds has been sharply reduced in China; and the number of lineages in the breeds has also been continuously decreased, and genetic diversity is lowered, so that the pig breeds are on the edge of extinction in many places. In recent years, with the improvement of consciousness of protecting local breed resources, the local pig breeds have been protected by taking a variety of measures, while cryopreserving boar semen is a convenient and effective preservation means. Freezing of semen is a semen source with a higher bio-safety coefficient. The frozen semen may be reserved when idle so as to balance the supply and demand. Since fresh semen has short preservation time, the demand of the semen has exceeded supply in rutting season, but the semen has been wasted due to excess production capacity when idle. Through freezing of the semen, qualified semen can be totally cryopreserved, and unqualified semen is removed in time, thereby improving semen quality and balancing demands in low season and peak season. The semen may also be allocated across regions at any time so as to balance the demands in different production regions and achieve the advantage of convenience in transport. The cost can be saved; efficiency may be increased; and feeding breeding stock of sires is lowered. A frozen semen integration technology may increase a utilization rate of the male animals to be close to 100%, greatly decrease the feeding breeding stock
Description
of sires, save the cost of field construction, feeding and management, decrease construction lands, decrease waste emissions and relieve the environmental protection pressure, and is also an important means of breed conservation and breeding. The frozen semen has a characteristic of long-term preservation, and may prolong the usage time of genetic materials of the sires. It is not limited by death of the sires and the like, and has important significances in aspects of reserving and restoring semen supply, updating the lineages and introducing the breeds. The frozen semen is always an important means of breed conservation and breeding. In the late 1980s, after lots of foreign pig breeds were introduced into China, pig farmers bred the foreign pigs for pursuit of high meat yield and short-term economic benefits, and the number of bred local pigs was sharply declined. At the present stage, key technical researches on frozen production and preservation of porcine semen have been taken into account and concerned by global pig industry. Although the researches on porcine frozen semen in China has made some breakthroughs in technology, lots of researches report that, production of the frozen semen of pigs, particularly local pig breeds, needs to be further improved technically. Due to biological characteristics of sperms of the local pig breeds, the freezing and production technology of the semen of the local pig breeds is required to be not exactly the same as a production technology of the frozen semen of commercial pigs; and the sperms of the local pig breeds are easily damaged in freezing and unfreezing processes. Therefore, the key technical problems of pig breeding include: deeply and systematically researching a freezing mechanism of the porcine semen and a key technology in the production process, investigating the factors that influence vitality and fertilization ability of porcine frozen semen and further improving the freezing technology and operating method of the porcine semen, and particularly urgent problems in breeding production of national local pig breeds need to be solved.
Description
Summary A purpose of the present invention is to provide a base diluent formula for porcine frozen semen and a preparation method thereof. Another purpose of the present invention is to provide a specific application of the formula, for overcoming the defects in the prior art. To achieve the above purposes, technical solutions of the present invention are as follows: A base diluent for porcine frozen semen includes glucose, citric acid, cholesterol, L-proline, Tris and double distilled water. Further, the cholesterol is preferably cholesterol from pork liver. Further, the base diluent for porcine frozen semen includes 15.98+0.5 g/L of glucose, 19.670.5 g/L of citric acid, 4.83+0.2 g/L of cholesterol, 3.45+0.2 g/L of L-proline, 35.20.5 g/L of Tris and 1 L of double distilled water. In addition to the above base diluent for porcine frozen semen, a cryoprotectant of the porcine frozen semen further includes the following components in percentage by volume: 20% of fresh egg yolk, 1x106 IU/L of penicillin, 1x106 IU/L of streptomycin, 3-4% of glycerin and 1-2% of Equex.STM. In addition to the above base diluent for porcine frozen semen, a porcine preservation semen diluent further includes isotonic skimmed milk, wherein a volume ratio of the amount of the isotonic skimmed milk to the base diluent for porcine frozen semen is 1:1; and a 0.22 pm bacterial filter membrane is used for filtration. Further, the isotonic skimmed milk is commercially available skimmed milk. A preparation method of the base diluent for porcine frozen semen includes the steps: weighing the glucose, the citric acid and the Tris; taking the cholesterol from pork liver, the L-proline and the double distilled water; mixing the materials
Description
in a beaker; heating the mixture to completely dissolve the materials; and cooling the mixture. A preparation method of the cryoprotectant of the porcine frozen semen includes the steps: weighing the glucose, the citric acid and the Tris; taking the cholesterol from pork liver, the L-proline and the double distilled water; mixing the materials in a beaker; heating the mixture to completely dissolve the materials; cooling the mixture; adding the fresh egg yolk, the penicillin and the streptomycin into the diluent; uniformly mixing the materials and cooling; standing and precipitating the liquid; centrifuging the supernatant; removing a surface oil film of the centrifuged diluent; and adding glycerin and Equex.STM, thereby obtaining the cryoprotectant. Further, in the above preparation method, the materials are uniformly mixed and cooled to 4°C; the liquid was stood and precipitated for 8-12 h; and solid impurities in the egg yolk can be fully settled in the steps. After the supernatant is centrifuged at 3850-4000 rpm for 90 min, surface oil impurities are absorbed by a ml pipettor; and the oil impurities in the egg yolk can be completely separated onto the surface of the diluent in the step. A preparation method of the porcine preservation semen diluent includes the steps: weighing the glucose, the citric acid and the Tris; taking the cholesterol from pork liver, the L-proline and the double distilled water; mixing the materials in a beaker; heating the mixture to 98°C for completely dissolving the materials; cooling the mixture to 35°C; and mixing the mixture with isotonic skimmed milk according to a volume ratio of 1:1, thereby obtaining the porcine preservation semen diluent. A method for preparing porcine frozen semen by utilizing the base diluent for porcine frozen semen includes the following steps: (1) collecting semen: collecting porcine middle-piece semen; filtering the semen with filter paper; and measuring density during semen collection, wherein
Description
semen having original semen density of less than 100 million/ml is directly eliminated; and semen having the original semen density of more than 100 million/ml is adjusted and diluted; (2) diluting: mixing the semen collected in the step (1) with the porcine preservation semen diluent according to a volume ratio of 1:1; and balancing the mixture at 17°C, wherein the porcine preservation semen diluent provides sufficient energy and nutrients; and sperm viability can be maintained to the full extent; (3) centrifugal suspension: centrifuging the mixed semen diluent obtained in the step (2); removing the supernatant; adding the cryoprotectant of the porcine frozen semen into a precipitate; and standing the liquid for 10-15 min to enable sperms with excellent viability to come up, to further eliminate sperms with poor viability and dead sperms; (4) filling; (5) refrigeration balancing: balancing the filled semen in a balancing tank at 4°C for 3-4 h, to increase stability and make preparations for freezing; (6) freezing: measuring the viability (>90%) and freezing in sequence after balancing is ended; and freezing the semen by a freezing curve. Further, after the step (2), the method further includes steps: analyzing the sperm viability; slightly and uniformly mixing the semen added with the diluent; then measuring the viability; and directly abandoning the sperms with poor viability, so as to achieve an effect of maintaining the viability of the transported sperms to be more than 90%. Further, in the step (3), centrifugal conditions are as follows: a rotation speed is 1850 rpm; and time is 10 min. Further, in the step (6), the freezing curve used in the freezing process shall not excessively deviate; and the freezing curve is as follows: a starting temperature
Description
of 5°C, -6°C for 200 s, -6°C for 60 s, -100°C for 160 s, -140°C for 80 s, and -140°C for 180 s. An unfreezing method based on the porcine frozen semen includes steps: adding the frozen semen into a water bath at 37°C for 20 s; incubating the semen for 10 min; and detecting viability of the semen. The base diluent for porcine frozen semen and the cryoprotectant and the preservation semen diluent prepared based on the diluent can be applied to preparing the porcine frozen semen, particularly applied to preparing frozen semen of local pig breeds in China. Certainly, the semen freezing technology suitable for the local pig breeds is also applicable to semen freezing technologies of imported pig breeds and other pig breeds. Advantages and technical effects of the present invention are as follows: In the present invention, a survival rate (the survival rate can be up to 95% or higher) of fresh semen of the local pig breeds during long-distance transport can be effectively maintained; and by adding the cholesterol from pork liver, the L-proline and the antifreeze agent Equex.STM, damage of the porcine sperms in the freezing process can be prevented or significantly decreased. Moreover, through the effective unfreezing method, the operation is convenient; and the sperm viability and quality are improved (the highest viability may be up to 90% after unfreezing). The semen diluent and the cryoprotectant in the present invention are simple in preparation; the method is convenient to operate and low in cost; and the high-quality porcine frozen semen can be obtained.
Detailed Description
The present invention will be further described below in combination with embodiments. Embodiment 1:
Description
A preparation method of a base diluent for porcine frozen semen includes the steps: glucose, citric acid and Tris were weighed; cholesterol from pork liver, L-proline and double distilled water were taken; the materials were mixed in a beaker; the mixture was heated to completely dissolve the materials; and the mixture was cooled. 1.598 g of glucose, 1.967 g of citric acid, 3.52 g of Tris, 0.483 g of cholesterol from pork liver, 0.345 g of L-proline and 100 ml of double distilled water may be weighed. A preparation method of a cryoprotectant of the porcine frozen semen includes the steps: the glucose, the citric acid and the Tris were weighed first; the cholesterol from pork liver, the L-proline and the double distilled water were mixed in a beaker; the mixture was heated to completely dissolve the materials; the mixture was cooled; then 20% of fresh egg yolk, penicillin and streptomycin were added into the diluent; the materials were uniformly mixed and cooled to 4°C; the liquid was stood and precipitated for 12 h; the supernatant was centrifuged at 3850 rpm for 90 min; the centrifuged supernatant was taken; and 4% of glycerin and 1% of antifreeze agent Equex.STM (Nova Chemical Sales, Scituate, MA, USA) were added, thereby obtaining the cryoprotectant. A preparation method of a porcine preservation semen diluent includes the steps: the glucose, the citric acid and the Tris were weighed; the cholesterol from pork liver, the L-proline and the double distilled water were mixed in a beaker; the mixture was heated to 98°C for completely dissolving the materials; the mixture was cooled to 35°C; and the mixture was mixed with isotonic skimmed milk according to a volume ratio of 1:1, thereby obtaining the porcine preservation semen diluent. A method for preparing porcine frozen semen by utilizing the base diluent for porcine frozen semen includes the following steps:
'7
Description
(1) collecting semen: porcine middle-piece semen was collected; the semen was filtered with filter paper; and density was measured during semen collection, wherein semen having original semen density of less than 100 million/ml was directly eliminated; and semen having the original semen density of more than 100 million/ml was adjusted and diluted; (2) diluting: the semen collected in the step (1) was mixed with the porcine preservation semen diluent according to a volume ratio of 1:1; and the mixture was balanced at 17C, wherein the porcine preservation semen diluent provided sufficient energy and nutrients; and sperm viability can be maintained to the full extent; the semen added with the diluent was slightly and uniformly mixed; then the viability was measured; and the sperms with poor viability were directly abandoned, to achieve an effect of maintaining the viability of the transported sperms to be more than 90%; (3) centrifugal suspension: the mixed semen diluent obtained in the step (2) was centrifuged (at a rotation speed of 1850 rpm for 10 min); the supernatant was removed; the cryoprotectant was added into a precipitate; and the liquid was stood for 10-15 min to enable sperms to come up, to further eliminate sperms with poor viability and dead sperms; (4) filling; (5) refrigeration balancing: the filled semen was balanced in a balancing tank at 4°C for 3-4 h, to increase stability and make preparations for freezing; (6) freezing: measurement of the viability (more than 90%) and freezing were conducted in sequence after balancing was ended; and the semen was frozen by a freezing curve, wherein the freezing curve shall not excessively deviate; and the freezing curve was as follows: a starting temperature of 5°C, -6°C for 200 s, -6°C for 60 s, -100°C for 160 s, -140°C for 80 s, and -140°C for 180 s. An unfreezing method based on the porcine frozen semen includes steps:
Description
the frozen semen was added into a water bath at 37°C for 20 s; the semen was incubated for 10 min; and viability of the unfrozen semen was detected. Embodiment 2: Local pig breeds used in the present embodiment are Laiwu black pig, Dapulian pig and Licha black pig. Experiments were conducted based on the cryoprotectant of the porcine frozen semen, the preservation semen diluent and the preparation methods in embodiment 1. 1. Preparation of the cryoprotectant of the porcine frozen semen Egg yolk liquid was prepared the day before semen collection; estimation was conducted according to the number of animals collected in the next day; and frozen semen of 200-300 pieces of 0.5 mL tubes can be prepared after one ejaculation of one local pig. The cryoprotectant was prepared based on the base diluent for porcine frozen semen (see embodiment 1 for details). 2. Preparation of preservation semen diluent The preservation semen diluent was prepared the day before semen collection; based on the base diluent for porcine frozen semen (1.598 g of glucose, 1.967 g of citric acid, 3.52 g of Tris, 0.483 g of cholesterol from pork liver, 0.345 g of L-proline and 100 ml of double distilled water were taken; the materials were added into an appropriate beaker; the mixture was heated to 98°C for completely dissolving the materials; the mixture was cooled to 35°C; and the mixture was mixed with isotonic skimmed milk according to a ratio of 1:1, thereby obtaining the porcine preservation semen diluent), the semen and the preservation semen diluent shall be mixed according to a volume ratio of 1:1; and generally, the prepared diluent was further filtered by a 0.22 pm bacterial filter membrane, thereby preventing impurities from affecting the vitality of sperms. 3. Semen collection and dilution
Description
Semen was collected (note: a semen collection frequency was 2-3 times per week, once every 2-3 days); middle-piece semen was generally collected; the collected semen shall be filtered with filter paper; the semen was balanced at 17°C for 25-30 min; the semen was mixed with the preservation semen diluent according to a ratio of 1:1 (balanced with fresh semen at 17°C simultaneously); and the semen was transported at 17°C (for 3-4 h). 4. Density measurement and analysis of sperm vitality 2-3 mL of original semen was collected during semen collection; density of the original semen shall be measured by a full-automatic sperm density meter when the semen arrived at a frozen semen production workshop; the original semen was uniformly mixed; the middle-piece semen was absorbed and measured; semen having original semen density of less than 100 million/ml was directly eliminated; and semen having the original semen density of more than 100 million/ml was adjusted and diluted. The sperm vitality was analyzed by combining automatic phase contrast microscope analysis software with a microscope; the semen added with the diluent was slightly and uniformly mixed; then the vitality was measured; a glass slide and a cover glass shall be preheated in advance; the sperms were prevented from being stimulated by an extremely low temperature; and the vitality of the transported sperms may be up to 95% or higher. 5. Centrifugal suspension A rotation speed of a centrifuge was set at 1850 rpm; time was set as 10 min; the mixed semen diluent was centrifuged; the supernatant was absorbed after centrifugation; 5-6 mL of the cryoprotectant was added into a precipitate; and the liquid was stood for 10-15 min to enable sperms to come up. 6. Egg yolk dilution The sperms of the same variety that came up were mixed in a 50 mL sterilized centrifuge tube; vitality of the sperms was observed under the microscope; density
1 )
Description
of the sperms was adjusted (100 million/ml); and if the density was too high, the cryoprotectant was added to dilute the semen. A variety number of the treated semen shall be marked on the centrifuge tube to prevent the treated semen from being mixed with other varieties. 7. Full-automatic filling printing The semen with adjusted density was subjected to packing printing (0.5 mL tube) on a full-automatic filling printer; variety numbers and ear numbers were printed; and the variety numbers, the dates and the animal ear numbers were printed on the tubes. 8. Tube racking The filled tubes of the same variety and ear number were placed on a tube rack; and the tubes of different varieties and ear numbers are separately racked. 9. Refrigeration balancing 1-2 tubes were reserved for each variety and ear number and put into a disc while filling printing, thereby facilitating vitality detection after balancing. The tubes were put into a balancing tank at 4°C for balancing for 3-4 h. 10. Full-automatic program freezing 10.1 A liquid nitrogen container, a pail, a foam box, thumb tubes, sterile gauze bags (having a length of about 15 cm and a width of about 5 cm), filaments, tweezers, label paper and scissors were prepared before freezing; the pail was put into the foam box to be filled with liquid nitrogen for later use. 10.2 After balancing was ended, the viability (>90%) was measured, and freezing was started; the 0.5 mL tube was taken out of the balancing tank and then put into a frozen semen tank according to the above freezing curve; the semen shall be frozen for about 10 min; the freezing process shall be performed according to the freezing curve; and excessive deviation shall be avoided. 10.3 The tube was rapidly placed after the freezing was ended. 11. Tube filling
Description
The 0.5 mL tubes were filled into the thumb tubes; then the thumb tubes were filled into the sterile gauze bags; each bag was filled with ten thumb tubes; a rope was tied to two bags; information (such as variety numbers, animal ear numbers, accurate tube number, dates and unit name) shall be labeled on the bags and label paper; the label paper was attached to a position that was 30-40 cm above the end of the rope; the sterile gauze bags were put into the liquid nitrogen container; and samples were pressed by the pail, so that the samples were completely immersed into the liquid nitrogen. 12. Measurement of the viability The viability of the frozen semen prepared in the week was detected once every week; whether the viability reached the standard was observed; and the semen was eliminated in time if the viability did not reach the standard. The viability was measured again when the semen was separated and discharged from storehouse, so as to ensure that the semen is qualified. Unfreezing method: Through multiple repeated experiments, it was finally determined that, the unfreezing method included: the tubes were placed in a water bath at 37°C for 20 s; then the tubes were cut off; the semen was incubated in a centrifuge tube for 10 min; and the viability of the semen was detected. Result analysis: The formulas such as the base diluent for porcine frozen semen and the cryoprotectant in the present invention and the porcine tube frozen semen prepared by the procedures provided by the present invention are taken as experimental groups; porcine tube frozen semen prepared by the existing porcine tube frozen semen preparation kit in the market is taken as a control group; and data comparison of the experimental groups and the control group after unfreezing is as follows:
1*')
Description
Table 1 Comparison of unfreezing effects of tube frozen semen of pigs (Licha black pigs) Group Number of Original Average vitality after Average aberration rate
experiments semen vitality unfreezing after unfreezing
Control group 100 290% 48.69+3.21 % 16.29+4.18
% Experimental group 100 290% 80.35+4.07aa % 13.87+2.65
% (Licha black pigs)
Notes: the superscript a represents that the experimental group and the control group have significant differences; the superscript aa represents that the experimental group and the control group have extremely significant differences; and no superscript represents that the experimental group and the control group have no significant difference. Table 2 Comparison of unfreezing effects of tube frozen semen of pigs (Laiwu black pigs) Group Number of Original Average vitality after Average aberration rate
experiments semen vitality unfreezing after unfreezing
Control group 100 >90% 35.87±3.42 % 18.31±3.98
% Experimental group 100 >90% 78.72±2.13aa % 14.19±1.13 % (Laiwu black pig)
Notes: the superscript a represents that the experimental group and the control group have significant differences; the superscript aa represents that the experimental group and the control group have extremely significant differences; and no superscript represents that the experimental group and the control group have no significant difference. Table 3 Comparison of unfreezing effects of tube frozen semen of pigs (Dapulian pigs) Group Number of Original Average vitality after Average aberration rate
experiments semen vitality unfreezing after unfreezing
Control group 100 285% 28.71+4.05 % 13.87+3.06 %
Experimental group 100 285% 75.35+5.76aa % 15.90+3.11 %
(Dapulian black pig)
Description
Notes: the superscript a represents that the experimental group and the control group have significant differences; the superscript aa represents that the experimental group and the control group have extremely significant differences; and no superscript represents that the experimental group and the control group have no significant difference. Results of the experiments in Tables 1-3 show that, the experimental group of the unfreezing vitality of tube frozen semen of the local pig breeds (Licha black pigs) and the control group have extremely significant difference; and results of the other two local pig breeds (i.e., the Laiwu black pigs and the Dapulian pigs) are consistent with experimental results of the Licha black pigs. The transported porcine fresh semen with preservation semen diluent in the present invention is taken as an experimental group; the transported porcine fresh semen with existing porcine fresh semen diluent in the market is taken as a control group; and data comparison of fresh semen vitality in the experimental group and the control group after 3-4 hours from long-distance transport is as follows: Table 4 Comparison of long-distance transport effects of fresh semen of pigs (Licha black pigs) Group Number of Original Average vitality after Average aberration rate
experiments semen vitality transport after transport
Control group 30 290% 69.32+2.01 % 3.19+1.28% % Experimental group 30 290% 93.25+2.87aa 2.14+0.93%
(Licha black pigs)
Notes: the superscript a represents that the experimental group and the control group have significant differences; the superscript aa represents that the experimental group and the control group have extremely significant differences; and no superscript represents that the experimental group and the control group have no significant difference. Table 5 Comparison of long-distance transport effects of fresh semen of pigs (Laiwu black pigs)
1A
Description
Group Number of Original Average vitality after Average aberration experiments semen vitality transport rate after transport
Control group 30 >90% 71.90±4.11 % 5.51±0.63%
Experimental group 30 >90% 90.09±5.71aa % 6.45±1.07
% (Laiwu black pigs)
Notes: the superscript a represents that the experimental group and the control group have significant differences; the superscript aa represents that the experimental group and the control group have extremely significant differences; and no superscript represents that the experimental group and the control group have no significant difference. Table 6 Comparison of long-distance transport effects of fresh semen of pigs (Dapulian black pigs) Group Number of Original Average vitality after Average aberration experiments semen vitality transport rate after transport
Control group 30 >85% 59.31±4.31 % 5.73±1.14
% Experimental group 30 >85% 90.25±5.76aa % 6.89±0.36
% (Dapulian black pigs)
Notes: the superscript a represents that the experimental group and the control group have significant differences; the superscript aa represents that the experimental group and the control group have extremely significant differences; and no superscript represents that the experimental group and the control group have no significant difference. Results of the experiments in Tables 4-6 show that, the experimental group of the fresh semen of the local pig breeds (Licha black pigs) after long-distance transport and the control group have extremely significant differences; and results of the other two local pig breeds (i.e., the Laiwu black pigs and the Dapulian pigs) are consistent with experimental results of the Licha black pigs. It can be seen from the analysis of the above experimental results that, compared with the prior art, the base diluent formula for porcine frozen semen, the cryoprotectant for the porcine frozen semen, the porcine preservation semen
Description
diluent, and the preparation and unfreezing methods of the porcine frozen semen provided by the present invention can significantly increase the vitality of the porcine semen and significantly decrease the aberration rate of the semen, i.e., the quality of the porcine frozen semen can be effectively improved by the present invention.
1A
Claims (10)
1. A base diluent for porcine frozen semen, comprising glucose, citric acid, cholesterol, L-proline, Tris and double distilled water.
2. The base diluent for porcine frozen semen according to claim 1, wherein the cholesterol is cholesterol from pork liver.
3. A cryoprotectant of the porcine frozen semen, further comprising egg yolk, penicillin, streptomycin, glycerin and Equex.STM based on the base diluent for porcine frozen semen of claim 1 or 2.
4. A porcine preservation semen diluent, further comprising isotonic skimmed milk based on the base diluent for porcine frozen semen of claim 1 or 2, wherein a volume ratio of the amount of the isotonic skimmed milk to the base diluent for porcine frozen semen is 1:1.
5. The base diluent for porcine frozen semen according to claim 1, wherein a preparation method of the base diluent for porcine frozen semen comprises the steps: weighing the glucose, the citric acid and the Tris; taking the cholesterol from pork liver, the L-proline and the double distilled water; mixing the materials in a beaker; heating the mixture to completely dissolve the materials; and cooling the mixture.
6. A preparation method of the cryoprotectant of the porcine frozen semen of claim 3, comprising the steps: weighing the glucose, the citric acid and the Tris; taking the cholesterol from pork liver, the L-proline and the double distilled water; mixing the materials in a beaker; heating the mixture to completely dissolve the materials; cooling the mixture; adding fresh egg yolk, penicillin and streptomycin into the diluent; uniformly mixing the materials and cooling; standing and precipitating the liquid; centrifuging the supernatant; removing a surface oil film of the centrifuged diluent; and adding the glycerin and the Equex.STM, thereby obtaining the cryoprotectant.
7. A method for preparing porcine frozen semen based on claims 1-4, comprising the following steps:
Claims
collecting semen: collecting porcine middle-piece semen; filtering the semen with filter paper; and measuring density during semen collection, wherein semen having original semen density of less than 100 million/ml is directly discarded; and semen having the original semen density of more than 100 million/ml is adjusted and diluted; diluting: mixing the semen collected in the step (1) with the porcine preservation semen diluent according to a volume ratio of 1:1; and balancing the mixture at 17C, wherein the porcine preservation semen diluent provides sufficient energy and nutrients; and sperm viability can be maintained to the full extent; centrifugal suspension: centrifuging the mixed semen diluent obtained in the step (2); removing the supernatant; adding the cryoprotectant of the porcine frozen semen into a precipitate; and standing the liquid for 10-15 min to enable sperms with excellent viability to come up, to further eliminate sperms with poor viability and dead sperms; filling; refrigeration balancing: balancing the filled semen in a balancing tank at 4°C for 3-4 h to increase stability and make preparations for freezing; freezing: measuring the viability (>90%) and freezing in sequence after balancing is ended; and freezing the semen by a freezing curve.
8. The method according to claim 7, wherein in the step (3), centrifugal conditions are as follows: a rotation speed is 1850 rpm and time is 10 min; and in the step (6), the freezing curve is as follows: a starting temperature of 5°C, -6°C for 200 s, -6°C for 60 s, -100°C for 160 s, -140°C for 80 s, and -140°C for 180 s.
9. An unfreezing method for the porcine frozen semen of claim 7, comprising steps: adding the porcine frozen semen into a water bath at 37°C for 20 s; incubating the semen for 10 min; and detecting viability of the semen after unfreezing.
Claims
10. An application of the base diluent for porcine frozen semen of claim 1 in preparation of frozen semen of local pig breeds.
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