CN105941391B - A kind of hybridization snakehead spermatozoa preservative fluid and its application - Google Patents
A kind of hybridization snakehead spermatozoa preservative fluid and its application Download PDFInfo
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- CN105941391B CN105941391B CN201610353178.3A CN201610353178A CN105941391B CN 105941391 B CN105941391 B CN 105941391B CN 201610353178 A CN201610353178 A CN 201610353178A CN 105941391 B CN105941391 B CN 105941391B
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The present invention provides a kind of hybridization snakehead spermatozoa preservative fluids, using water as solvent, are preserved in liquid per 1L and contain following components：Sodium chloride, 0.4 ~ 0.5g potassium chloride, 0.1 ~ 0.15g calcium chloride, 0.2 ~ 0.6g sodium bicarbonates, the 8 ~ 15g glucose of 7.5 ~ 8.5g, surplus is water.Hybridization snakehead spermatozoa preservative fluid provided by the invention source is simple, it is easy to operation, it preserves using conventional low temperature, after preserving the activation of spermatozoa that liquid preserves, long lifespan is preserved, activity ratio is high, and sperm motility is good, sperm storage and Reproduction to hybridize snakehead provide guarantee, have preferable application prospect.
Description
Technical field
The invention belongs to fish culture technical fields, more particularly, to a kind of hybridization snakehead spermatozoa preservative fluid and its answer
With.
Background technology
Hybridization snakehead belongs to Perciformes, murrel suborder, murrel section, Ophiocephalus on taxology, and Guangdong is commonly called as raw fish, is fresh water ferociousness
Predacious fish.Hybridize snakehead delicious flavour, full of nutrition, is extensively received by the market.In recent years, the kind mainly cultivated in the market
It is snakehead（♂）With the snakehead belonged to（♀）Hybridize the hybridized snakehead fish generated.Snakehead, snakehead are the fish that lay eggs in batches, in hybridized snakehead fish
In reproductive process, often face that female, milter sexal maturity is asynchronous or the phenomenon of female adult degree deficiency.In order to avoid sperm wave
Take, Cord blood is carried out to sperm, it is very necessary.In general, cryopreservation is carried out with liquid nitrogen, the sperm storage time is more
Length, vigor are stronger.But for most of aquatic products seedlings enterprise, acquisition and storage liquid nitrogen are inconvenient, and sperm is super
It is more demanding to freezing, recovery program and relevant parameter etc. technology when Cord blood, it is not easy to grasp.In contrast, conventional
Low temperature（4℃）Preservation only needs general refrigerator, and function definition is had more in production practices.
Invention content
It is an object of the invention to according to the deficiency in existing fish sperm Techniques of preserving, provide a kind of hybridization snakehead essence
Son preserves liquid.
Another object of the present invention is to provide application of the above-mentioned preservation liquid in hybridizing snakehead sperm storage.
Snakehead milter and this kind can be preferably solved according to preservation liquid provided by the invention or belong to raun artificial propagation
When, the nonsynchronous problem of gonad development, and face in production sperm rapid deactivation the problem of.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The present invention provides a kind of hybridization snakehead spermatozoa preservative fluids, using water as solvent, are preserved per 1L in liquid containing with the following group
Point：The sodium chloride of 7.5 ~ 8.5g, 0.4 ~ 0.5g potassium chloride, 0.1 ~ 0.15g calcium chloride, 0.2 ~ 0.6g sodium bicarbonates, the Portugals 8 ~ 15g
Grape sugar, surplus is water.
Preferably, 1L, which is preserved in liquid, contains following components：The sodium chloride of 8.2g, 0.47g potassium chloride, 0.128g calcium chloride,
0.4g sodium bicarbonates, 10g glucose, surplus are water；
The osmotic pressure for preserving liquid is 343 mOsm/kg, pH value 7.4.
Snakehead sperm and the preservation liquid will be hybridized according to 1：9 proportioning is diluted, and is placed at 4 DEG C and is preserved.It preserves
After 96h sperm be averaged quick run duration be 177.4 seconds, entire life be 206.3 seconds, activity ratio 94.7%；Essence after preservation 120h
The average quick run duration of son is 141.2 seconds, and entire life is 158.4 seconds, activity ratio 82.5%.
Compared with prior art, the invention has the advantages that：
Hybridization snakehead spermatozoa preservative fluid provided by the invention source is simple, easy to operation, preserves using the low of routine
Temperature preserves long lifespan after preserving the activation of spermatozoa that liquid preserves, and activity ratio is high, and sperm motility is good, for hybridization snakehead
Sperm storage and Reproduction provide guarantee, have preferable application prospect.
Specific implementation mode
Further illustrated the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagent, methods and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.
Embodiment 1：
It is accurate to weigh sodium chloride 8.2g, potassium chloride 0.47g, calcium chloride 0.128g, sodium bicarbonate 0.4g, glucose 10g, it is molten
Solution is configured to spermatozoa preservative fluid in 1 liter of distilled water（Total osmotic pressure is 343 mOsm/kg, pH value 7.4）.
Embodiment 2：
Sodium chloride 8.5g, potassium chloride 0.5g, calcium chloride 0.15g, sodium bicarbonate 0.6g, glucose 14g accurately are weighed, is dissolved
In 1 liter of distilled water, it is configured to spermatozoa preservative fluid（Total osmotic pressure is 380mOsm/kg, adjusts pH value to 7.4）.
Comparative example 1：
It is accurate weigh saleratus 10g, sodium dihydrogen phosphate 10g, sucrose 42.7g, reduced glutathione 2g,（Always ooze
Pressure is 664mOsm/kg thoroughly, adjusts pH value to 7.4）.It is dissolved in 1 liter of distilled water, is configured to spermatozoa preservative fluid.
Comparative example 2：
Accurately weigh sodium chloride 6.6g, potassium chloride 0.15g, calcium chloride 0.15g, sodium bicarbonate 0.2g, sodium dihydrogen phosphate
0.1g is dissolved in 1 liter of distilled water, is configured to spermatozoa preservative fluid.（Total osmotic pressure is 242mOsm/kg, adjusts pH value to 7.4）.
Comparative example 3：
Sodium chloride 7.5g, potassium chloride 0.2g, calcium chloride 0.2g, sodium bicarbonate 10.2g accurately are weighed, is dissolved in 1 liter of distillation
In water, it is configured to spermatozoa preservative fluid.（Total osmotic pressure is 272mOsm/kg, adjusts pH value to 7.4）.
Embodiment 3:Sperm motility measures
Snakehead sperm will be hybridized and preserve liquid according to 1：9 proportioning is diluted, and is placed at 4 DEG C and is preserved.Sperm is at 4 DEG C
After being preserved 96 hours in lower preservation liquid, its vigor and run duration are measured.
Sperm motility measures in embodiment and comparative example：Sperm after 0.5ml dilutes is added dropwise on common glass slide, is added
0.5ml water activates, and observes its vigor under 10 × 16 power microscopes immediately.The results are shown in Table 1：
Table 1
| Quick run duration（s） | Entire life（s） | Activity ratio（%） | |
| Embodiment 1 | 177.4 | 206.3 | 94.7 |
| Embodiment 2 | 152.7 | 189.4 | 90.4 |
| Comparative example 1 | 127.3 | 140.2 | 83.7 |
| Comparative example 2 | 135.6 | 167.8 | 85.9 |
| Comparative example 3 | 110.8 | 130.5 | 81.4 |
It is best to sperm storage effect to can be seen that the spermatozoa preservative fluid being formulated in embodiment 1 from result in table 1.
We continue the sperm motility measurement of spermatozoa preservative fluid in embodiment 1 after preserving 120h simultaneously：It preserves
After 120h sperm be averaged quick run duration be 141.2 seconds, entire life be 158.4 seconds, activity ratio 82.5%.
Embodiment 4：
1, sexually matured male hybridization snakehead body surface wiped clean will be had reached, abdomen is splitted, takes out spermary, and careful
Surface fat is rejected with tweezers in ground, weighs on clean balance.
Spermary is placed in clean quartzy mortar, is shredded, ground with scissors, be slowly added into 1 ~ 49 times of volume in batches
Embodiment 1 in spermatozoa preservative fluid（Specifically it is shown in Table 2）, then filtered into clean beaker with 80 mesh yarn thin,tough silk, then it is sub-packed in
Centrifuge tube is placed in 4 DEG C of refrigerators and saves backup.
2, preservation effect is evaluated
Sperm number measures：The dilution sperm that Conventional cryogenic is preserved takes out, and is placed at room temperature.0.2ml sperm is taken to be placed in
In biological tally counting frame, in 40 × 16 times of optical microphotographs microscopic observations, 8 chosen in 16 big frame record its sperm
Number.It is computed, after preserving 96h, sperm number is 13.9 × 109A/ml accounts for the 92% of fresh essence；After preserving 120h, sperm number
It is 11.9 × 109A/ml accounts for the 79% of fresh essence.
Meanwhile we also have detected the vigor of sperm 12h under different dilution ratios in preserving liquid, the results are shown in Table 2：
Table 2
| Dilution ratio | Quick run duration (RMT) | Service life (LS) sec | Activity ratio (AR) % |
| Control group | 147.36±1.11 | 175.42±0.97 | 87.45±2.09 |
| 1:1 | 141.61±1.63 | 167.36±1.51 | 80.89±0.42 |
| 1:4 | 144.56±2.95 | 175.19±1.09 | 82.50±0.53 |
| 1:9 | 178.91±1.86 | 209.20±2.14 | 94.82±0.22 |
| 1:19 | 166.72±0.93 | 196.73±1.15 | 89.08±0.45 |
| 1:49 | 162.33±2.21 | 188.65±2.48 | 87.92±0.20 |
Note：Control group is not add preservation liquid.
As a result it shows：Dilution ratio is 1:When 9, preservation effect is best.
Claims (3)
1. a kind of hybridization snakehead spermatozoa preservative fluid, which is characterized in that 1L, which is preserved in liquid, contains following components：The sodium chloride of 8.2g,
0.47g potassium chloride, 0.128g calcium chloride, 0.4g sodium bicarbonates, 10g glucose, surplus are water；
The osmotic pressure for preserving liquid is 343 mOsm/kg, pH value 7.4.
2. the application described in claim 1 for preserving liquid in hybridizing snakehead sperm storage.
3. application according to claim 2, which is characterized in that by hybridization snakehead sperm and the preservation liquid according to 1:9
Proportioning is diluted, and is placed at 4 DEG C and is preserved.
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| CN110419471B (en) * | 2019-09-05 | 2021-11-16 | 中国水产科学研究院淡水渔业研究中心 | Method for artificial insemination of wild burmese reeves shad and hatching of fertilized eggs |
| CN115362961B (en) * | 2022-04-14 | 2023-12-08 | 中国水产科学研究院北戴河中心实验站 | Artificial insemination method of Songjiang perch and application thereof |
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| CN104521943A (en) * | 2014-12-09 | 2015-04-22 | 南京师范大学 | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen |
| CN104604845A (en) * | 2015-02-06 | 2015-05-13 | 中国水产科学研究院黑龙江水产研究所 | Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method |
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|---|---|---|---|---|
| CN104521943A (en) * | 2014-12-09 | 2015-04-22 | 南京师范大学 | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen |
| CN104604845A (en) * | 2015-02-06 | 2015-05-13 | 中国水产科学研究院黑龙江水产研究所 | Hucho taimen seminal fluid ultralow-temperature cryopreservation fluid and hucho taimen seminal fluid freezing and preserving method |
Non-Patent Citations (2)
| Title |
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| 中华乌塘鳢精子的生物学;江世贵 等;《水产学报》;20000430;第24卷(第2期);第119-122页 * |
| 乌鳢精子生理生态特性的研究Ⅱ钠、钾及渗透压对精子活力的影响;赵振山等;《水利渔业》;19951231(第3期);第13-15页 * |
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