CN102986650A - Finless eel sperm diluent and preparation method thereof - Google Patents
Finless eel sperm diluent and preparation method thereof Download PDFInfo
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- CN102986650A CN102986650A CN2012105535780A CN201210553578A CN102986650A CN 102986650 A CN102986650 A CN 102986650A CN 2012105535780 A CN2012105535780 A CN 2012105535780A CN 201210553578 A CN201210553578 A CN 201210553578A CN 102986650 A CN102986650 A CN 102986650A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a finless eel sperm diluent and a preparation method thereof. The finless eel sperm diluent is prepared from sodium chloride, potassium chloride, sodium bicarbonate, calcium chloride, and sodium hydrogen phosphate according to a ratio. The preparation method comprises the steps of A, preparing sperm diluent mother liquor: preparing sperm diluent mother liquors respectively containing 1mmol of sodium chloride, 1mmol of potassium chloride, 1mmol of sodium bicarbonate, 0.5mmol of calcium chloride, and 0.5mmol of sodium hydrogen phosphate in per ml, and storing the mother liquors in a refrigerator, wherein the mother liquor containing sodium hydrogen phosphate is prepared by the steps of calculating the weight of sodium hydrogen phosphate for preparing the mother liquor according to the concentration and volume of the mother liquor and the molecular weight of sodium hydrogen phosphate, weighing sodium hydrogen phosphate, and putting sodium hydrogen phosphate into a beaker; and B, preparing the sperm diluent: measuring and preparing the diluent from the mother liquor containing each component, evenly mixing each component, evenly mixing the prepared diluent in a flask, diluting to a certain volume by distilled water, and storing the prepared diluent in the refrigerator for use, wherein the amount of each mother liquor is calculated according to the concentration of each component in the formulation of the diluent and the volume of the diluent. The finless eel sperm diluent is reasonable in formulation, convenient to use, and suitable for diluting the finless eel sperm; the sperm is effectively diluted; the artificial insemination cost is reduced, and the fertility rate is improved.
Description
Technical field
The present invention relates to the sperm technical field of swamp eel, more specifically relate to a kind of swamp eel spermatozoa diluent, also relate to simultaneously a kind of preparation method of swamp eel spermatozoa diluent, be fit to all swamp eel kinds.
Background technology
Swamp eel (Monopterus albus) is the subtropics fish, has another name called yellow eel, sieve fish, field eel, Changyu, yellow ginseng, without squama son, Tian Can etc., is under the jurisdiction of Symbranchir, closes gill section; The whole world only has a kind, i.e. swamp eel in the Symbranchir.Be distributed widely in Japan, Thailand, Indonesia, Philippine, Malaysia, India, Japan, Korea and the China etc. in Southeast Asia, all there is distribution in China except the Qinghai-Tibet Platean, especially with In Middle And Lower Reaches of Changjiang River, distribution density is large, output is high, is a kind of important economic fish.
In order further to carry out the breeding of new variety of swamp eel, and the artificial propagation of swamp eel, for market provides more swamp eel new varieties and the seed of high-quality, need to carry out the swamp eel sperm super-low temperature and preserve and artificial propagation.Yet, no matter be the ultralow temperature preservation of sperm, still carry out artificial propagation, all need a kind of dilution that the swamp eel sperm is diluted.This is because the density of contained sperm is very high in the swamp eel seminal fluid, generally can reach 1.4 ~ 1.8 * 10
9Individual/ml.When the swamp eel sperm being carried out the ultralow temperature preservation, the smart stoste that contains the high density sperm can not directly be carried out the superfreeze preservation.Because in refrigerating process, when temperature drops to 0 ℃ ~-20 ℃ when interval, can form ice crystal in the seminal fluid that is saved, the sperm around ice crystal stabs as sharp cutter punctures sperm.Sperm concentration in the seminal fluid is higher, and the probability that sperm is punctured is also higher.This failure that ice crystal will directly cause sperm super-low temperature freezing to be preserved to the physical damnification of sperm in refrigerating process.Therefore, must dilute sperm before freezing, this just need to have a kind of spermatozoa diluent of suitable swamp eel sperm.And when carrying out artificial propagation, because it is very high to contain the density of sperm in the swamp eel seminal fluid, and sperm volume seldom, if directly be used for carrying out artificial insemination, be a kind of waste to very valuable seminal fluid not only, and, because sperm concentration is too high, the stickiness of seminal fluid increases, and makes sperm be difficult for mixing with ovum, thereby causes Insemination effect also bad.If after seminal fluid diluted, be used for again carrying out artificial insemination, can greatly reduce test-tube cost, can also play good Insemination effect simultaneously, obtain higher fertilization rate.Therefore, the effect of swamp eel spermatozoa diluent is exactly under the prerequisite that does not activate sperm, and sperm is diluted, and makes sperm keep as much as possible its original biological property.
Yet spermatozoa diluent is to have certain requirements.The sperm of every kind of fish has own certain osmotic pressure, and the osmotic pressure of fish of the same race is not different.The sperm of fish is not in resting state all the time before meeting water or other solution, freshwater fish only just can be activated when meeting the solution that is lower than own osmotic pressure or fresh water, and seawater fish just can be activated in the time of then will meeting the solution that is higher than own osmotic pressure or seawater.In a single day sperm is activated, and death very soon can decay.The life-span of sperm is generally very short, and short only has tens seconds, and is long also with regard to dozens of minutes.Want sperm is diluted, will guarantee that at first sperm can not be activated in dilution.This just requires the spermatozoa diluent of every kind of fish to equate as much as possible with the seminal fluid osmotic pressure of this fish.The osmotic pressure of dilution is to determine that by the ion concentration summation in the dilution dilution of variable concentrations just has different osmotic pressure, and the ion concentration of regulating in the dilution just can be regulated the osmotic pressure of this dilution, thereby reaches the effect of oozing with seminal fluid etc.In addition, different ion components has again different effects to sperm in the dilution.As: the Na ion in the dilution mainly has been the effect of regulating dilution osmotic pressure; The K ion plays a part to change sperm membrane surface permeability; Ca, Mg ion then play a part to increase or reduce sperm motility speed; And CO
3Ion then plays a part to regulate dilution pH etc.In addition, large minute subclass solutes such as reporting demonstration glucose is arranged except playing the effect of regulating osmotic pressure, also have the effect that the sperm energy is provided.Therefore, different fish not only diluent concentration is fully different, and according to the biological property of sperm, its ion component also has very big-difference.
Summary of the invention
The objective of the invention is to be to provide a kind of swamp eel spermatozoa diluent, prescription rationally, and is easy to use, and suitable dilution to the swamp eel sperm is the preservation of swamp eel sperm super-low temperature and carry out the necessary a kind of solution of artificial insemination.In swamp eel sperm super-low temperature preservation process, can guarantee that sperm can not be activated, and reduce to greatest extent ice crystal to the damage of sperm.In carrying out swamp eel artificial insemination process, can effectively dilute sperm, reduce the artificial insemination cost, improve fertilization rate.
Another object of the present invention is the preparation method who has been to provide a kind of swamp eel spermatozoa diluent, and is easy to implement the method, easy and simple to handle, is fit to the dilution of the various volumes of preparation, can guarantee the accuracy of a concentration of component in institute's prepared and diluted liquid.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
A kind of swamp eel spermatozoa diluent, it is made by the raw material of following weight portion:
Dilution osmotic pressure is 326.6 ~ 390.2mOsM, and pH is 7.5-8.5;
A kind of swamp eel spermatozoa diluent, it makes (better scope) by the raw material of following weight portion:
Dilution osmotic pressure is 343.7 ~ 383.1mOsM, and pH is 7.6-8.4.
A kind of swamp eel spermatozoa diluent, it makes (preferable range) by the raw material of following weight portion:
Dilution osmotic pressure is 349.9 ~ 376.9mOsM, and pH is 7.7-8.3.
A kind of swamp eel spermatozoa diluent, it makes (optimum range) by the raw material of following weight portion:
Dilution osmotic pressure is 356.6 ~ 370.8mOsM, and pH is 7.8-8.2.
A kind of swamp eel spermatozoa diluent, it makes (optimum value) by the raw material of following weight portion:
The dilution osmotic pressure of this prescription is 363.4mOsM, and pH is 8.0.
A kind of preparation method of swamp eel spermatozoa diluent the steps include:
1. the preparation of spermatozoa diluent mother liquor:
(1) mother liquor of every milliliter of sodium chloride-containing 1mmol: concentration, volume and sodium chloride molecular weight according to mother liquor calculate the weight sodium chloride of preparing the mother liquor needs, and counting accuracy is 0.0g.Accurately take by weighing the pure sodium chloride of this gravimetric analysis (NaCl) with balance, sodium chloride is put into beaker, add volume and be about the distilled water that needs are prepared mother liquor volume 1/2nd, sodium chloride is fully dissolved, sodium chloride solution after the dissolving is poured in the volumetric flask that needs the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.NaCl weight computing formula is as follows:
Solute weight=(mother liquid concentration * molecular weight solute) ÷ mother liquor volume
Following solute weight is all calculated by this formula.
(2) mother liquor of every milliliter of chloride containing potassium 1mmol: concentration, volume and potassium chloride molecular weight according to mother liquor calculate the potassium chloride weight of preparing the mother liquor needs, and counting accuracy is 0.0g.Accurately take by weighing the pure potassium chloride of this gravimetric analysis (KCl) with balance, potassium chloride is put into beaker, add volume and be about the distilled water that needs are prepared mother liquor volume 1/2nd, potassium chloride is fully dissolved, Klorvess Liquid after the dissolving is poured in the volumetric flask that needs the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(3) every milliliter of mother liquor that contains sodium bicarbonate 1mmol: concentration, volume and sodium bicarbonate molecular weight according to mother liquor calculate the sodium bicarbonate weight of preparing the mother liquor needs, and counting accuracy is 0.0g.Accurately take by weighing the pure sodium bicarbonate (NaHCO of this gravimetric analysis with balance
3), sodium bicarbonate is put into beaker, add volume and be about the distilled water that needs are prepared mother liquor volume 1/2nd, sodium bicarbonate is fully dissolved, sodium bicarbonate solution after the dissolving is poured in the volumetric flask that needs the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(4) mother liquor of every milliliter of chloride containing calcium 0.5mmol: concentration, volume and calcium chloride molecular weight according to mother liquor calculate the calcium chloride weight of preparing the mother liquor needs, and counting accuracy is 0.0g.Accurately take by weighing the pure calcium chloride (CaCl of this gravimetric analysis with balance
2), calcium chloride is put into beaker, add volume and be about the distilled water that needs are prepared mother liquor volume 1/2nd, calcium chloride is fully dissolved, calcium chloride solution after the dissolving is poured in the volumetric flask that needs the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(5) mother liquor of every milliliter of sodium hydrogen phosphate 0.5mmol: concentration, volume and calcium chloride molecular weight according to mother liquor calculate the sodium hydrogen phosphate weight of preparing the mother liquor needs, and counting accuracy is 0.0g.Accurately take by weighing the pure sodium hydrogen phosphate (Na of this gravimetric analysis with balance
2HPO
4), sodium hydrogen phosphate is put into beaker, add volume and be about the distilled water that needs are prepared mother liquor volume 1/2nd, sodium hydrogen phosphate is fully dissolved, disodium phosphate soln after the dissolving is poured in the volumetric flask that needs the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(6) above-mentioned mother liquor is all placed 4 ℃ of Refrigerator stores, and is for subsequent use.
2. the preparation of spermatozoa diluent:
(1) with microscale sampler (specification: each the volume required component of accurate measuring prepared and diluted liquid from each component mother liquor of 5 μ l ~ 5ml).Measure the required volume of each component according to concentration, the concentration of each component mother liquor and the dilution volume of required preparation of each component in the dilution prescription, be calculated as follows:
Measure volume=(formula concentration * dilution volume) ÷ mother liquid concentration
(2) above-mentioned each component (sodium chloride, potassium chloride, sodium bicarbonate, calcium chloride, sodium hydrogen phosphate, the dilution osmotic pressure of this prescription is 363.4mOsM, pH is 7.5-8.5) is mixed.If need the dilution volume of preparation greater than 100ml, then then mixing in the volumetric flask of respective volume is settled to the volumetric flask scale with distilled water.If need the dilution volume of preparation less than 100ml, mixing in vial then, then the dilution volume of preparation deducts each component volume summation of having measured as required, needs exactly the distilled water volume (because the minimum 100ml of being of volumetric flask) that adds.
(3) dilution for preparing is placed 4 ℃ of Refrigerator stores, and is for subsequent use.
Comparison with other fish sperm dilutions:
Following table is the comparison of swamp eel sperm and other several fish sperm diluent ingredients
The comparison of swamp eel spermatozoa diluent and other fish sperm dilutions has the following advantages and effect:
1. there are larger difference in the composition of swamp eel spermatozoa diluent and concentration and other fish sperm dilution.The swamp eel spermatozoa diluent belongs to the ionic dilution that mineral salt form, and the dilution that belongs to this class together has carp spermatozoa diluent and Sperm of Anguilla Japonica dilution.But with carp spermatozoa diluent ratio, many CaCl in the swamp eel spermatozoa diluent
2And Na
2HPO
4Composition.And with Sperm of Anguilla Japonica dilution ratio, the swamp eel spermatozoa diluent has lacked again MgCl
2Composition.
2. also there is larger difference in the osmotic pressure of swamp eel spermatozoa diluent with the osmotic pressure of other fish sperm.The swamp eel spermatozoa diluent belongs to height and oozes dilution.Its osmotic pressure is far above the hypotonic dilution of mandarin sturgeon, and the while is a little less than the dilution osmotic pressure of southern catfish.With it approaches common eel and silver carp spermatozoa diluent arranged.
3. although the pH of swamp eel spermatozoa diluent is basic identical with some fish, as: common eel, mandarin sturgeon.But still there is certain difference with the pH of other fish.
Embodiment
Embodiment 1:
Below preserve 10ml swamp eel seminal fluid as example take ultralow temperature (196 ℃), preparation and the using method of swamp eel spermatozoa diluent is described.
A kind of swamp eel spermatozoa diluent, it is made by the raw material of following weight portion:
The dilution osmotic pressure of this prescription is 371.7mOsM, and pH is 8.2.
A kind of preparation method of swamp eel spermatozoa diluent the steps include:
1. the preparation of spermatozoa diluent mother liquor:
(1) preparation 1000ml concentration is the NaCl mother liquor of 1M: according to the computing formula of describing in the method, calculate the weight sodium chloride that need to take by weighing.Accurately take by weighing the analytically pure NaCl of 58.4g with balance, be positioned in the 1000ml beaker, add about 500ml distilled water, after NaCl fully dissolves, be poured in the 1000ml volumetric flask, wash beaker 3 times with 5 ~ 10ml distilled water at every turn, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(2) preparation 500ml concentration is the KCl mother liquor of 1M: according to the computing formula of describing in the method, calculate the potassium chloride weight that need to take by weighing.Accurately take by weighing the analytically pure KCl of 37.3g with balance, be positioned in the 500ml beaker, add about 250ml distilled water, after KCl fully dissolves, be poured in the 500ml volumetric flask, wash beaker 3 times with 5 ~ 10ml distilled water at every turn, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(3) preparation 500ml concentration is the NaHCO of 1M
3Mother liquor: according to the computing formula of describing in the method, calculate the sodium bicarbonate weight that need to take by weighing.Accurately take by weighing the analytically pure NaHCO of 84.0g with balance
3, be positioned in the 500ml beaker, add about 250ml distilled water, treat NaHCO
3Fully after the dissolving, be poured in the 500ml volumetric flask, use 5 ~ 10ml distilled water washing beaker 3 times at every turn, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(4) preparation 500ml concentration is the CaCl of 0.5M
2Mother liquor: according to the computing formula of describing in the method, calculate the calcium chloride weight that need to take by weighing.Accurately take by weighing the analytically pure CaCl of 55.5g with balance
2, be positioned in the 500ml beaker, add about 250ml distilled water, treat CaCl
2Fully after the dissolving, be poured in the 500ml volumetric flask, use 5 ~ 10ml distilled water washing beaker 3 times at every turn, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(5) preparation 500ml concentration is the Na of 0.5M
2HPO
4Mother liquor: according to the computing formula of describing in the method, calculate the sodium hydrogen phosphate weight that need to take by weighing.Accurately take by weighing the analytically pure Na of 179.0g with balance
2HPO
4, be positioned in the 500ml beaker, add about 250ml distilled water, treat Na
2HPO
4Fully after the dissolving, be poured in the 500ml volumetric flask, use 5 ~ 10ml distilled water washing beaker 3 times at every turn, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last.
(6) above-mentioned mother liquor is all placed 4 ℃ of Refrigerator stores, and is for subsequent use.
2.100ml the preparation of spermatozoa diluent:
(1) according to formula: measure volume=(formula concentration * dilution volume) ÷ mother liquid concentration, calculate each the component volume that from mother liquor, to measure.Concrete volume is seen data in (2).
(2) the NaCl mother liquor of difference accurate measuring 16.8ml 1M; The KCl mother liquor of 680 μ l 1M; 530 μ l NaHCO
3The mother liquor of 1M; The CaCl of 340 μ l0.5M
2Mother liquor; The Na of 140 μ l0.5M
2HPO
4Mother liquor is in the 100ml volumetric flask.
(3) with behind the mentioned solution mixing, adding distil water is settled to 100ml.
(4) the above-mentioned 100ml swamp eel spermatozoa diluent for preparing is placed 4 ℃ of Refrigerator stores, for subsequent use.
3. the ultralow temperature of swamp eel sperm (196 ℃) is preserved:
(1) collection of swamp eel sperm: select sexual gland fully-developed swamp eel milter 5 ~ 10 tails, dry gonopore on every side and the water of belly with dry towel.Gently push the milter belly with have gentle hands, after extruding seminal fluid, then several seminal fluid in front are not clamp-oned seminal fluid in the dry container.Behind the seminal fluid mixing of extruding, prepare microscopy.
(2) sperm viability detects: carry out microscopy at 10 * 20 times microscopicallies, the bright smart average motility rate of computer-aided detection reaches more than 85%, averaged curve movement velocity (VCL) reaches more than 35 little meter per seconds, average point-to-point speed reaches more than 20 little meter per seconds, and this sperm just can be used for superfreeze and preserves.Put into 4 ℃ of Refrigerator stores, for subsequent use.
(3) dilution of seminal fluid and balance: get the seminal fluid that meets freezing standard behind the 10ml microscopy, add the swamp eel spermatozoa diluent that 30ml prepares in advance, thinner ratio is 1:3.Behind seminal fluid and the dilution mixing, place 4 ℃ of refrigerators, froze forward horizontal stand 20 minutes ~ 2 hours.
(4) add antifreeze and packing: the seminal fluid of above-mentioned dilution balance after 20 minutes added 3.2ml methyl-sulfoxide (DMSO), its final concentration is reached about 8%, immediately packing behind the mixing.With the 2ml microsyringe above-mentioned mixed liquor is sucked in the 2.0ml cryovial.Be divided into and fill 20 cryovials, then cryovial is placed on the freezing frame, per 5 cryovials are put 1 freezing frame, totally 4 freezing frames.Should prepare the bubble chamber of 1 about 30cm * 30cm * 20cm before the packing, put into trash ice in the case, aforesaid operations is all finished at the ice face, and the time can not be above 1 hour 40 minutes.After packing is finished, immediately freezing frame is put into the cool chamber of programmed cooling instrument, prepared cooling.
(5) programmed cooling: cooling process is down to-15 ℃ since 0 ℃ with 5 ℃/minute speed, stops 5 minutes at-15 ℃, and then is down to-85 ℃ with 25 ℃ of/minute speed from-15 ℃, locates balance 10 minutes at-85 ℃ at last.After cooling was finished, the freezing frame that straw directly will be housed took out from the programmed cooling instrument, puts into immediately liquid nitrogen container, preserves in-196 ℃ of temperature.
Embodiment 2-6
Its preparation method embodiment 1 is identical.
Embodiment 7:
Below take the ovum of artificial insemination 20 tail Buddhist nun swamp eel rauns as example, the using method of swamp eel dilution in the artificial insemination process is described.
A kind of swamp eel spermatozoa diluent, it is made by the raw material of following weight portion:
The dilution osmotic pressure of this prescription is 358.2mOsM, and pH is 7.9.
A kind of preparation method of swamp eel spermatozoa diluent the steps include:
1. the preparation of spermatozoa diluent mother liquor:
Spermatozoa diluent mother liquor compound method and case one are identical.
2.20ml the preparation of spermatozoa diluent:
(1) according to formula: measure volume=(formula concentration * dilution volume) ÷ mother liquid concentration, calculate each the component volume that from mother liquor, to measure.Concrete volume is seen data in (2).
(2) the NaCl mother liquor of difference accurate measuring 3.26ml 1M; The KCl mother liquor of 126 μ l 1M; The NaHCO of 96 μ l1M
3Mother liquor; The CaCl of 56 μ l 0.5M
2Mother liquor; The Na of 24 μ l 0.5M
2HPO
4Mother liquor is in 30ml vial bottle.
(3) with behind the mentioned solution mixing, add 16.44ml distilled water.
(4) the above-mentioned 20ml swamp eel spermatozoa diluent for preparing is placed 4 ℃ of Refrigerator stores, for subsequent use.
3. the artificial insemination of swamp eel
(1) collection of swamp eel sperm: select sexual gland fully-developed swamp eel milter 1 ~ 2 tail, dry gonopore on every side and the water of belly with dry towel.Gently push the milter belly with have gentle hands, after extruding seminal fluid, then several seminal fluid in front are not clamp-oned seminal fluid in the dry container.Behind the seminal fluid mixing of extruding, put into 4 ℃ of Refrigerator stores, for subsequent use.
(2) dilution of seminal fluid and balance: get the 5ml seminal fluid with dry sampler, add the swamp eel spermatozoa diluent that 10ml prepares in advance, thinner ratio is 1:2.Behind seminal fluid and the dilution mixing, place 4 ℃ of refrigerators, balance is more than 1 hour.
(3) swamp eel ovum artificial collection: the seminal fluid after to be diluted is after the refrigerator balance is about 1 hour, select the female swamp eel of 20 tail gonadal maturations, wipe fundatrigenia fish gonopore on every side and belly with dry towel respectively, according to pushing gently belly from head to the tail direction, the ovum of this 20 tail raun is all clamp-oned in the dry container, and normal temperature is for subsequent use.
(4) swamp eel artificial insemination: the 15ml seminal fluid that balance is good is poured in the ovum, stirs evenly gently with feather.Add the swamp eel activation of spermatozoa liquid that 250 ~ 300ml prepares in advance (the in addition issue of its prescription) after 1 ~ 2 minute, stirred gently 3 ~ 5 minutes, leave standstill 2 minutes after, add again 1000ml with supernatant water.After stirring gently 2 ~ 3 minutes again, outwell the sewage on the face, then wash ovum 2 ~ 3 times with this clear water, ovum can be moved in the incubator and hatch.
Claims (5)
1. swamp eel spermatozoa diluent, it is made by the raw material of following weight portion:
Parts by weight of raw materials
Sodium chloride 155.0-175.0mmol/L
Potassium chloride 5.5-7.5mmol/L
Sodium bicarbonate
4.0-6.0 mmol/L
Calcium chloride
1.0-2.0mmol/L
Sodium hydrogen phosphate
0.4-0.8mmol/L
Dilution osmotic pressure is 326.6 ~ 390.2mOsM, and pH is 7.5-8.5;
A kind of preparation method of swamp eel spermatozoa diluent the steps include:
The preparation of A, spermatozoa diluent mother liquor:
(1) mother liquor of every milliliter of sodium chloride-containing 1mmol: the weight sodium chloride that calculates preparation mother liquor needs according to concentration, volume and the sodium chloride molecular weight of mother liquor, take by weighing sodium chloride, sodium chloride is put into beaker, the distilled water that adds preparation mother liquor volume 1/2nd, sodium chloride is fully dissolved, sodium chloride solution after the dissolving is poured in the volumetric flask of the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last;
(2) mother liquor of every milliliter of chloride containing potassium 1mmol: the potassium chloride weight that calculates preparation mother liquor needs according to concentration, volume and the potassium chloride molecular weight of mother liquor, take by weighing potassium chloride, potassium chloride is put into beaker, the distilled water that adds mother liquor volume 1/2nd, potassium chloride is fully dissolved, Klorvess Liquid after the dissolving is poured in the volumetric flask of the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last;
(3) every milliliter of mother liquor that contains sodium bicarbonate 1mmol: the sodium bicarbonate weight that calculates the preparation mother liquor according to concentration, volume and the sodium bicarbonate molecular weight of mother liquor, take by weighing sodium bicarbonate, sodium bicarbonate is put into beaker, the distilled water that adds preparation mother liquor volume 1/2nd, sodium bicarbonate is fully dissolved, sodium bicarbonate solution after the dissolving is poured in the volumetric flask of the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last;
(4) mother liquor of every milliliter of chloride containing calcium 0.5mmol: the calcium chloride weight that calculates preparation mother liquor needs according to concentration, volume and the calcium chloride molecular weight of mother liquor, the calcium chloride that takes by weighing, calcium chloride is put into beaker, the distilled water that adds preparation mother liquor volume 1/2nd, calcium chloride is fully dissolved, calcium chloride solution after the dissolving is poured in the volumetric flask of the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last;
(5) mother liquor of every milliliter of phosphoric acid disodium hydrogen 0.5mmol: according to the concentration of mother liquor, volume and sodium hydrogen phosphate molecular weight calculate the sodium hydrogen phosphate weight of preparation mother liquor, take by weighing sodium hydrogen phosphate, sodium hydrogen phosphate is put into beaker, the distilled water that adds preparation mother liquor volume 1/2nd, sodium hydrogen phosphate is fully dissolved, disodium phosphate soln after the dissolving is poured in the volumetric flask of the same volume of preparation mother liquor, with No. 3 beakers of 5 ~ 10ml distilled water washing, also pour the distilled water after the washing into volumetric flask, be settled to volumetric flask scale place with distilled water at last;
(6) above-mentioned mother liquor is all placed 4 ℃ of Refrigerator stores;
The preparation of spermatozoa diluent:
(1) from each component mother liquor, measure prepared and diluted liquid with microscale sampler, measure the volume of each component according to the concentration of each component in the dilution prescription, the concentration of each component mother liquor and the dilution volume of preparation, be calculated as follows:
Measure volume=formula concentration * dilution volume ÷ mother liquid concentration;
(2) sodium chloride, potassium chloride, sodium bicarbonate, calcium chloride, each component of sodium hydrogen phosphate are mixed, the dilution volume 100ml of preparation, then mixing in the volumetric flask of volume is settled to the volumetric flask scale with distilled water;
(3) dilution for preparing is placed 4 ℃ of Refrigerator stores, and is for subsequent use.
2. a kind of swamp eel spermatozoa diluent according to claim 1 is characterized in that:
Parts by weight of raw materials
Sodium chloride 157.5-172.5mmol/L
Potassium chloride 5.75-7.25mmol/L
Sodium bicarbonate
4.25-5.75mmol/L
Calcium chloride
1.1-1.9mmol/L
Sodium hydrogen phosphate 0.45-0.75mmol/L
Dilution osmotic pressure is 343.7 ~ 383.1mOsM, and pH is 7.6-8.4.
3. a kind of swamp eel spermatozoa diluent according to claim 1 is characterized in that:
Parts by weight of raw materials
Sodium chloride 160.0-170.0mmol/L
Potassium chloride 6.0-7.0mmol/L
Sodium bicarbonate
4.5-5.5mmol/L
Calcium chloride
1.2-1.8mmol/L
Sodium hydrogen phosphate 0.5-0.7mmol/L
Dilution osmotic pressure is 349.9 ~ 376.9mOsM, and pH is 7.7-8.3.
4. a kind of swamp eel spermatozoa diluent according to claim 1 is characterized in that:
Parts by weight of raw materials
Sodium chloride 162.5-167.5mmol/L
Potassium chloride 6.25-7.0mmol/L
Sodium bicarbonate
4.75-5.25 mmol/L
Calcium chloride
1.3-1.7mmol/L
Sodium hydrogen phosphate 0.55-0.65mmol/L
Dilution osmotic pressure is 356.6 ~ 370.8mOsM, and pH is 7.8-8.2.
5. a kind of swamp eel spermatozoa diluent according to claim 1 is characterized in that:
Parts by weight of raw materials
Sodium chloride 165.0mmol/L
Potassium chloride 6.5mmol/L
Sodium bicarbonate
5.0 mmol/L
Calcium chloride
1.5mmol/L
Sodium hydrogen phosphate 0.6 mmol/L
The dilution osmotic pressure of this prescription is 363.4mOsM, and pH is 8.0.
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