CN111345283A - Method for freezing and storing buffalo semen - Google Patents
Method for freezing and storing buffalo semen Download PDFInfo
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- CN111345283A CN111345283A CN202010247033.1A CN202010247033A CN111345283A CN 111345283 A CN111345283 A CN 111345283A CN 202010247033 A CN202010247033 A CN 202010247033A CN 111345283 A CN111345283 A CN 111345283A
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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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Abstract
The invention relates to a frozen preservation method of buffalo semen, which comprises the steps of semen collection, dilution, freezing, preservation and the like, wherein the semen is collected by adopting a pseudo-vaginal method, the milky semen is selected, jelly is filtered and removed, and the semen with the quality up to the standard is preheated to 35 ℃; preheating the diluent to 35 ℃, diluting the semen, cooling the diluted semen to 4 ℃, subpackaging the diluted semen into 0.25mL thin tubes, sealing, and freezing, wherein the freezing procedure is as follows: the temperature is started from 4 ℃, the temperature is maintained at 4 ℃ for 30min, then the temperature is reduced to-10 ℃ at the speed of 1-5 ℃/min, then the temperature is reduced to-140 ℃ at the speed of 30-40 ℃/min from-10 ℃, finally the temperature is maintained at-140 ℃ for 10min, and the freezing is finished and the frozen. The method has simple steps and high practicability, and the thawing activity of the obtained frozen semen reaches 67.66 percent, which greatly exceeds the collection requirement of national-level livestock gene banks.
Description
Technical Field
The invention relates to a semen preservation method, in particular to a buffalo semen cryopreservation method, and belongs to the technical field of livestock breeding.
Background
The cattle in the upper sea are native to the suburbs of the upper sea such as Jiading, Baoshan, Fengxian and Chongming. The body size is large, the weight of a cow is 700-900 kg, and the weight of a bull exceeds 1000 kg. The buffalo is originally an important livestock used for both meat and meat in rice regions in south China, and the value of the buffalo in service is reduced according to the recorded records of 'livestock and poultry variety records in Shanghai city' in 1987, along with the development of agricultural mechanization and rural urbanization, the feeding amount of the buffalo in Shanghai is reduced at the speed of 0.13 head every year since 1972, only 2.89 thousands of buffalos exist in 1982, and the number of fine breed buffalos is sharply reduced. In the second livestock genetic resource survey of Shanghai buffalo in 2006, the first report is about being endangered, so that the Shanghai buffalo is not included in the 2012 edition of Chinese livestock genetic resource record.
The semen freezing preservation plays an important role in modern animal husbandry, the frozen semen can be used without the limitation of time, region and animal life, the breeding quantity of the male livestock is reduced, the production cost is reduced, the utilization rate of excellent livestock is improved, and the semen freezing preservation has extremely important significance on the introduction of the livestock, the updating of blood margins, the disease control, the endangered variety and the protection of genetic diversity. The present Chinese frozen bovine semen production process is implemented by referring to the technical specification of the production of frozen bovine semen of the people's republic of China, but the recovery rate of the frozen semen is related to the quality of the original semen, the type of diluent, the semen freezing temperature, the thawing temperature and other factors, wherein the temperature change condition of the semen in the freezing process is one of the important factors influencing the semen freezing quality, the freezing procedure provided by freezing instrument and equipment manufacturers is adopted in the domestic production of frozen bovine semen, basically the same freezing procedure is used for the production of the frozen semen of various bovine products, and therefore, the survival rate of the frozen semen is lower after the frozen semen is thawed.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a method for freezing and preserving buffalo semen.
Technical scheme
A cryopreservation method of buffalo semen comprises the following steps:
(1) collection of semen
Selecting 2-6 years old adult male buffalo, adopting a pseudo-vaginal method to collect semen, selecting milky semen after semen collection, filtering to remove jelly, then performing quality inspection, and preheating the semen reaching the quality inspection to 35 ℃;
(2) dilution of semen
Preheating the diluent to 35 ℃, and then diluting the semen which reaches the standard in the quality inspection in the step (1) to obtain diluted semen;
(3) freezing and preservation of semen
Cooling the diluted semen to 4 ℃, filling the diluted semen into a 0.25mL thin tube by a canning machine at the temperature of 4 ℃, sealing, and then putting the thin tube into a program freezing instrument for freezing, wherein the freezing program is as follows: the temperature is started from 4 ℃, the temperature is maintained at 4 ℃ for 30min, then the temperature is reduced to-10 ℃ at the speed of 1-5 ℃/min, then the temperature is reduced to-140 ℃ at the speed of 30-40 ℃/min from-10 ℃, finally the temperature is maintained at-140 ℃ for 10min, the freezing is finished, and the frozen semen is put into liquid nitrogen for storage.
Further, in the step (1), the quality inspection comprises appearance inspection, sperm motility inspection and density inspection, the sperms with more floccules are required to be removed, and the sperm motility rate in the semen is more than 50%.
Further, in the step (2), the dilution method is as follows: the first step is as follows: diluting and uniformly mixing the semen and the diluent according to the volume ratio of 1:2 to obtain a mixed solution; the second step is that: and adding the equal volume of diluent into the mixed solution in the first step, and uniformly mixing to obtain diluted semen.
Further, in the step (3), the freezing procedure is as follows: the temperature is reduced to-10 ℃ at the speed of 1 ℃/min after the temperature is maintained at 4 ℃ for 30min, is reduced to-140 ℃ at the speed of 35 ℃/min from-10 ℃, and is finally maintained at-140 ℃ for 10 min.
The invention has the beneficial effects that: the invention provides a frozen preservation method of buffalo semen, which comprises the steps of firstly screening the semen with better quality, ensuring that the preserved semen has stronger viability, thereby being beneficial to improving the survival rate of the semen in the semen when the semen is diluted, then diluting the semen by two steps to reach the target viscosity, and finally reducing the diluted semen to the preservation temperature by an optimized freezing program, wherein the steps are simple, the practicability is high, the thawing activity of the frozen semen obtained by the method is averagely 44.1 percent and maximally 67.66 percent, and the thawing activity greatly exceeds the requirement of national livestock gene bank collection (the activity is more than or equal to 30 percent).
Detailed Description
The technical solution of the present invention will be described in detail with reference to the following specific examples.
In the following examples, the diluent is 2.5 Xconcentrated bovine semen diluent (Optidyl) produced by CRYO-VET of FranceTM) But is not limited thereto.
Example 1
A cryopreservation method of buffalo semen comprises the following steps:
(1) collection of semen
Selecting eight-head 2-6 year old adult Buffalo (Buffalo number: B18, A11, 3, E12, B12, 6, E11 and Z2) from Dongtai of Shanghai Chongming island, collecting semen by pseudo-vaginal method, selecting milky semen solution after semen collection, filtering with sterilized filter paper to remove jelly, performing quality inspection (eliminating semen with more floc, selecting semen with sperm activity rate of more than 50%), and preheating semen reaching quality inspection standard to 35 deg.C;
the conditions of the harvest amount are shown in table 1:
as can be seen from Table 1, the yield of 8 adult bulls used in the test is 1.0-7.2mL, the volume change is large, and the total yield in the second day is obviously superior to that in the first day.
(2) Dilution of semen
Preheating the diluent to 35 ℃, and then diluting the semen which reaches the quality inspection standard in the step (1) (in the first step, diluting and uniformly mixing the semen and the diluent according to the volume ratio of 1:2 to obtain a mixed solution; in the second step, adding the same volume of diluent into the mixed solution in the first step, and uniformly mixing to obtain the diluted semen;
(3) freezing and preservation of semen
Placing the diluted semen into a 4 ℃ thermostat, cooling to 4 ℃ in a water bath, filling into a 0.25mL thin tube by a filling machine at 4 ℃, sealing, and then placing into a program freezing instrument for freezing, wherein the freezing program is as follows: the temperature is reduced to-10 ℃ at the speed of 1 ℃/min after the temperature is maintained at 4 ℃ for 30min, then reduced to-140 ℃ at the speed of 35 ℃/min from-10 ℃, finally kept at-140 ℃ for 10min, the freezing is finished, and the frozen semen is put into liquid nitrogen for storage.
Semen thawing and conventional detection:
taking out the thin tube sample from the liquid nitrogen tank, quickly putting the thin tube sample into a 37 ℃ water bath kettle for thawing for 35s, taking 10 mu L of semen to drop in a sperm counting plate, putting the sperm counting plate on a 37 ℃ constant temperature objective table, randomly selecting 3 visual fields under 200 times of optical microscope by using a semen analyzer, and automatically determining the sperm motility rate and activity.
The experimental data are subjected to one-factor variance analysis by SPSS17.0 statistical software, the result is expressed as Mean plus or minus standard error (Mean plus or minus SEM), and the difference is obvious when P is less than 0.05. The results are shown in Table 2:
TABLE 2 sperm quality before and after freezing of seawater cattle
Note: the same letter between the same columns indicates no significant difference (P > 0.05); different letters indicate significant difference (P < 0.05).
As can be seen from table 2, the raw sperm motility rate (41.14%) of 8 adult buffalos collected in this example is higher than 50% except for the raw sperm motility rate (41.14%) of the buffalo with the number 6 and the raw sperm motility rate (49.87%) of the buffalo with the number Z2, and the raw sperm motility rate (88.43%) and the motility (86.42%) of the buffalo with the number B18 are the highest and are significantly higher than those of the other 7 adult buffalos (P < 0.05). The difference between the original sperm motility rate (58.59%) and the vitality (43.26%) of the buffalo with the number E12 is larger than that of other 7 buffalos.
As can be seen from Table 2, the difference in frozen semen quality between 8 adult buffalos (P <0.05) is significant, and the frozen-thawed buffalo semen with the number of B18 has the highest survival rate and activity, which are 76.24% and 67.66%, respectively. The number 3 buffalo has higher than 50% of original sperm motility, and the lowest sperm motility after unfreezing is 32.23% and 29.43%. The number 6 buffalo sperm with the lowest quality of original sperm had no significant decrease in post-freezing viability (41.24%) and motility (41.24%).
Example 2
The semen of the same batch of buffalo in example 1 is diluted and then frozen, and the freezing procedure is changed into: the temperature is started from 4 ℃, the temperature is maintained at 4 ℃ for 30min and then is reduced to-10 ℃ at the speed of 5 ℃/min, then the temperature is reduced to-140 ℃ at the speed of 40 ℃/min from-10 ℃, and finally the temperature is maintained at-140 ℃ for 10min, thus completing the freezing. The rest is the same as in example 1.
The results of sperm quality testing before and after buffalo freezing are shown in table 3:
table 3 example 2 sperm quality before and after freezing of marine cattle
Note: the same letter between the same columns indicates no significant difference (P > 0.05); different letters indicate significant difference (P < 0.05).
As can be seen from Table 3, the difference in frozen semen quality between 8 adult buffalos (P <0.05) is significant, the frozen-thawed survival rate and viability of the buffalo sperm numbered B18 are the highest, respectively, 66.40% and 62.65%, and the frozen survival rate of the buffalo numbered E11 is next to B18 and is 56.64%. The activity of the numbered buffalo semen A11 and Z2 after thawing was not significantly different from that of E11, and was 44.76% and 44.46% respectively. The number 3 buffalo had the lowest sperm motility, 39.45% and 30.45% after thawing.
Claims (4)
1. A frozen preservation method of buffalo semen is characterized by comprising the following steps:
(1) collection of semen
Selecting 2-6 years old adult male buffalo, adopting a pseudo-vaginal method to collect semen, selecting milky semen after semen collection, filtering to remove jelly, then performing quality inspection, and preheating the semen reaching the quality inspection to 35 ℃;
(2) dilution of semen
Preheating the diluent to 35 ℃, and then diluting the semen which reaches the standard in the quality inspection in the step (1) to obtain diluted semen;
(3) freezing and preservation of semen
Cooling the diluted semen to 4 ℃, filling the diluted semen into a 0.25mL thin tube by a canning machine at the temperature of 4 ℃, sealing, and then putting the thin tube into a program freezing instrument for freezing, wherein the freezing program is as follows: the temperature is started from 4 ℃, the temperature is maintained at 4 ℃ for 30min, then the temperature is reduced to-10 ℃ at the speed of 1-5 ℃/min, then the temperature is reduced to-140 ℃ at the speed of 30-40 ℃/min from-10 ℃, finally the temperature is maintained at-140 ℃ for 10min, the freezing is finished, and the frozen semen is put into liquid nitrogen for storage.
2. The method for cryopreservation of buffalo semen as claimed in claim 1, wherein in the step (1), the quality inspection comprises appearance inspection, sperm motility inspection and density inspection, and the sperm with more floccules is required to be removed, and the sperm motility rate in the semen is more than 50%.
3. The cryopreservation method of buffalo semen as claimed in claim 1, wherein in the step (2), the dilution method is as follows: the first step is as follows: diluting and uniformly mixing the semen and the diluent according to the volume ratio of 1:2 to obtain a mixed solution; the second step is that: and adding the equal volume of diluent into the mixed solution in the first step, and uniformly mixing to obtain diluted semen.
4. The cryopreservation method of buffalo semen as claimed in claim 1, 2 or 3, wherein in the step (3), the freezing procedure is as follows: the temperature is reduced to-10 ℃ at the speed of 1 ℃/min after the temperature is maintained at 4 ℃ for 30min, is reduced to-140 ℃ at the speed of 35 ℃/min from-10 ℃, and is finally maintained at-140 ℃ for 10 min.
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| CN202010247033.1A CN111345283B (en) | 2020-03-31 | 2020-03-31 | Method for freezing and storing buffalo semen |
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| CN202010247033.1A CN111345283B (en) | 2020-03-31 | 2020-03-31 | Method for freezing and storing buffalo semen |
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| CN111345283B CN111345283B (en) | 2022-02-15 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1927226A (en) * | 2006-09-11 | 2007-03-14 | 华中农业大学 | Cow sex-control sperm freezing preservation seminal fluid dilution |
| CN104542572A (en) * | 2013-10-22 | 2015-04-29 | 云南省种畜繁育推广中心 | Method for manufacturing straw frozen semen of gayal and application |
| CN106106433A (en) * | 2016-06-22 | 2016-11-16 | 湖北省畜禽育种中心 | A kind of Binglangjiang waterbuffalo freezing of semen process |
| CN107333752A (en) * | 2017-08-08 | 2017-11-10 | 重庆信首科技有限公司 | Kind ox seminal fluid Cryopreservation |
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2020
- 2020-03-31 CN CN202010247033.1A patent/CN111345283B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1927226A (en) * | 2006-09-11 | 2007-03-14 | 华中农业大学 | Cow sex-control sperm freezing preservation seminal fluid dilution |
| CN104542572A (en) * | 2013-10-22 | 2015-04-29 | 云南省种畜繁育推广中心 | Method for manufacturing straw frozen semen of gayal and application |
| CN106106433A (en) * | 2016-06-22 | 2016-11-16 | 湖北省畜禽育种中心 | A kind of Binglangjiang waterbuffalo freezing of semen process |
| CN107333752A (en) * | 2017-08-08 | 2017-11-10 | 重庆信首科技有限公司 | Kind ox seminal fluid Cryopreservation |
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