CN105494314A - Ancherythroculter nigrocauda sperm preservation method - Google Patents

Ancherythroculter nigrocauda sperm preservation method Download PDF

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CN105494314A
CN105494314A CN201510947925.1A CN201510947925A CN105494314A CN 105494314 A CN105494314 A CN 105494314A CN 201510947925 A CN201510947925 A CN 201510947925A CN 105494314 A CN105494314 A CN 105494314A
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sperm
distilled water
settled
cacl
nahco
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CN105494314B (en
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王贵英
李清
祝东梅
李佩
陈见
刘英武
蔡汉玲
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Wuhan Academy of agricultural science and technology
Wuhan Xianfeng Aquatic Product Science & Technology Co., Ltd.
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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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  • Engineering & Computer Science (AREA)
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Abstract

The invention discloses an ancherythroculter nigrocauda sperm preservation method. The ancherythroculter nigrocauda sperm preservation method comprises the steps that 1, a sperm preserving fluid is prepared: raw material components are weighed in proportion and are respectively dissolved by using distilled water and are mixed evenly, and the distilled water is added to achieve volume fixing; 2, sperm acquisition is performed: an adult ancherythroculter nigrocauda milter is toweled off, and urine and faeces are removed to avoid sperm pollution; the abdomen is pressed slightly, extruded-out sperm is collected by using a suction tube, and sunlight or ultraviolet irradiation is avoided in the acquisition process; 3, sperm storage is performed: acquired ancherythroculter nigrocauda sperm is arranged in a dry, clean and sealed container subjected to sterilization and disinfection, and the container containing the sperm and the preserving fluid is kept in a low-temperature light-avoiding mode after the acquisition is completed. By the adoption of the technical scheme, the in-vitro survival time of the ancherythroculter nigrocauda sperm can be effectively prolonged, the fertilization vitality of the sperm can be kept, and protective development and distant crossbreeding of ancherythroculter nigrocauda are achieved. The ancherythroculter nigrocauda sperm preservation method is low in cost, simple and short in process and easy to operate, enables the preserving time to be long and has important application value.

Description

A kind of nearly red Culter sperm storage method of black tail
Technical field
The present invention relates to sperm storage technical field, specifically a kind of nearly red Culter sperm storage method of black tail.
Background technology
The peculiar economic fish of black tail Jin HongCultershi China Upper Yangtze River, because overfishing, river construction are built, natural resources slump of disastrous proportions; Carry out artificial breeding to support, the Important Action being protection of resources and making full use of.In artificial propagation process, the nearly red Culter scale of black tail easily comes off injured, and not easy to operate, parent survival rate is low; It is shorter that the nearly red Culter sperm of black tail excretes the rear holding time, and as under 4 DEG C of conditions, can keep 80% survival rate in 3h, in 7h, survival rate is only 50%; In the nearly red Culter distant hybridization breeding process of black tail, because variety classes repoductive time there are differences, the efficient breeding time is short or to be hybridized to motility rate low.Utilize spermatozoa preservative fluid to preserve the nearly red Culter sperm of black tail, one is reduce parent's loss and consumption; Two is accurately grasp sperm viability, improves fertility rate and hatchability; Three is extend sperm storage time and distant hybridization breeding time, improves distant hybridization breeding survival rate, extensive cultivation new varieties or new lines.And there is holding time short and high in cost of production problem to sperm storage effect in existing spermatozoa preservative fluid.
Summary of the invention
The object of the present invention is to provide a kind of nearly red Culter sperm storage method of black tail, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
A kind of nearly red Culter sperm storage method of black tail, concrete steps are as follows:
(1) preparation of spermatozoa preservative fluid: take each material composition by proportioning, dissolve respectively with distilled water, mix, adding distil water is settled to 100mL;
Spermatozoa preservative fluid adopts the one in following formula to prepare:
A 1conserving liquid is filled a prescription: NaCl0.80g, KCl0.04g, CaCl 20.01g, MgSO 47H 2o0.02g, NaHCO 30.04g, Na 2hPO 4h 2o0.02g, KH 2pO 40.01g, C 6h 12o 6h 2o0.10g, distilled water is settled to 100mL;
A 2conserving liquid is filled a prescription: NaCl0.80g, KCl0.04g, CaCl 20.01g, MgSO 47H 2o0.02g, NaHCO 30.04g, Na 2hPO 4h 2o0.02g, KH 2pO 40.01g, C 6h 12o 6h 2o0.10g, penicillin 200 × 10 4iU, distilled water are settled to 100mL;
B 1conserving liquid is filled a prescription: NaCl0.7800g, KCl0.0200g, CaCl 20.0210g, NaHCO 30.0021g, distilled water are settled to 100mL;
B 2conserving liquid is filled a prescription: NaCl0.7800g, KCl0.0200g, CaCl 20.0210g, NaHCO 30.0021g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
C 1conserving liquid is filled a prescription: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, distilled water is settled to 100mL;
C 2conserving liquid is filled a prescription: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
D 1conserving liquid is filled a prescription: NaCl0.743g, KCl0.278g, CaCl 20.029g, MgCl 26H 2o0.030g, NaHCO 30.042g, distilled water is settled to 100mL;
D 2conserving liquid is filled a prescription: NaCl0.743g, KCl0.278g, CaCl 20.029g, MgCl 26H 2o0.030g, NaHCO 30.042g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
E 1conserving liquid is filled a prescription: NaCl0.6903g, KCl0.0946g, CaCl 20.0300g, MgCl 26H 2o0.0142g, Na 2hPO 40.0540g, NaHCO 30.3999g, TYR 0.0996g, glycine 0.0412g, bovine serum albumin(BSA) 0.4110g, distilled water is settled to 100mL;
E 2conserving liquid is filled a prescription: NaCl0.6903g, KCl0.0946g, CaCl 20.0300g, MgCl 26H 2o0.0142g, Na 2hPO 40.0540g, NaHCO 30.3999g, TYR 0.0996g, glycine 0.0412g, bovine serum albumin(BSA) 0.4110g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
(2) semen collection: towel off the nearly red Culter milter of dry ripe black tail and get rid of urine and ight soil, avoiding seminal fluid to pollute; Light pressure belly, collect the seminal fluid extruded with suction pipe, gatherer process avoids sunlight or Ultraviolet radiation;
(3) seminal fluid is deposited: be placed in by near for the black tail of collection red Culter seminal fluid through the drying of sterilization, cleaning and the container of sealing, the volume ratio of seminal fluid and spermatozoa preservative fluid is 1:5 ~ 10, gathers after terminating and the container filling seminal fluid and conserving liquid is put 3 ~ 5 DEG C of low-temperature darks preservations.
As the further scheme of the present invention: the preferred version of the nearly red Culter spermatozoa preservative fluid of black tail is conserving liquid formula C 2: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, penicillin 200 × 10 4iU, distilled water is settled to 100mL, under 4 DEG C of conditions, conserving liquid formula C 2the holding time making the nearly red Culter sperm viability of black tail reach 80% is 156h.
Compared with prior art, the invention has the beneficial effects as follows: the present invention, by above technical scheme, effectively can extend the black tail nearly red Culter sperm in vitro time-to-live, keep its fertilization vigor, realizes protective development and the distant hybridization breeding of the nearly red Culter of black tail.The method is with low cost, briefly easy to operate, and effective holding time is long, has important using value.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Preserve vigor for improving the nearly red Culter sperm in vitro of black tail, realize protective development, its spermatozoa preservative fluid each composition distilled water is formulated.
(1) preparation of spermatozoa preservative fluid: take each material composition by proportioning, dissolve respectively with distilled water, mix, adding distil water is settled to 100mL;
Spermatozoa preservative fluid adopts the one in following formula to prepare:
A 1conserving liquid is filled a prescription: NaCl0.80g, KCl0.04g, CaCl 20.01g, MgSO 47H 2o0.02g, NaHCO 30.04g, Na 2hPO 4h 2o0.02g, KH 2pO 40.01g, C 6h 12o 6h 2o0.10g, distilled water is settled to 100mL;
A 2conserving liquid is filled a prescription: NaCl0.80g, KCl0.04g, CaCl 20.01g, MgSO 47H 2o0.02g, NaHCO 30.04g, Na 2hPO 4h 2o0.02g, KH 2pO 40.01g, C 6h 12o 6h 2o0.10g, penicillin 200 × 10 4iU, distilled water are settled to 100mL;
B 1conserving liquid is filled a prescription: NaCl0.7800g, KCl0.0200g, CaCl 20.0210g, NaHCO 30.0021g, distilled water are settled to 100mL;
B 2conserving liquid is filled a prescription: NaCl0.7800g, KCl0.0200g, CaCl 20.0210g, NaHCO 30.0021g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
C 1conserving liquid is filled a prescription: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, distilled water is settled to 100mL;
C 2conserving liquid is filled a prescription: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
D 1conserving liquid is filled a prescription: NaCl0.743g, KCl0.278g, CaCl 20.029g, MgCl 26H 2o0.030g, NaHCO 30.042g, distilled water is settled to 100mL;
D 2conserving liquid is filled a prescription: NaCl0.743g, KCl0.278g, CaCl 20.029g, MgCl 26H 2o0.030g, NaHCO 30.042g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
E 1conserving liquid is filled a prescription: NaCl0.6903g, KCl0.0946g, CaCl 20.0300g, MgCl 26H 2o0.0142g, Na 2hPO 40.0540g, NaHCO 30.3999g, TYR 0.0996g, glycine 0.0412g, bovine serum albumin(BSA) 0.4110g, distilled water is settled to 100mL;
E 2conserving liquid is filled a prescription: NaCl0.6903g, KCl0.0946g, CaCl 20.0300g, MgCl 26H 2o0.0142g, Na 2hPO 40.0540g, NaHCO 30.3999g, TYR 0.0996g, glycine 0.0412g, bovine serum albumin(BSA) 0.4110g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
(2) semen collection: towel off the nearly red Culter milter of dry ripe black tail and get rid of urine and ight soil, avoiding seminal fluid to pollute; Light pressure belly, collect the seminal fluid extruded with suction pipe, gatherer process avoids sunlight or Ultraviolet radiation;
(3) seminal fluid is deposited: be placed in by near for the black tail of collection red Culter seminal fluid through the drying of sterilization, cleaning and the container of sealing, the volume ratio of seminal fluid and spermatozoa preservative fluid is 1:5 ~ 10, gathers after terminating and the container filling seminal fluid and conserving liquid is put 3 ~ 5 DEG C of low-temperature darks preservations.
The optimal case of the nearly red Culter spermatozoa preservative fluid of black tail is conserving liquid formula C 2: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, penicillin 200 × 10 4iU, distilled water is settled to 100mL, under 4 DEG C of conditions, conserving liquid formula C 2the holding time making the nearly red Culter sperm viability of black tail reach 80% is 156h, and the penicillin added can extend the holding time of the nearly red Culter sperm of black tail.
Sperm storage effect: the vigor (rapid movement sperm accounts for the percentage of total number of sperm) after being activated with sperm is evaluated preservation effect for index.First the spermatozoa preservative fluid preserving sperm is taken out during observation and shake up, then be placed on cleaning, dry slide with liquid-transfering gun and disposable tip sucking-off one droplet, sperm is observed under microscope 16 × 10 multiple, activate sperm with half distilled water immediately after observing sperm, observe its motion conditions and record.Add 2.0 × 10 4the conserving liquid of IU/mL penicillin can obviously extend the sperm storage time; Conserving liquid C 2preservation effect best, conserving liquid E 2take second place, conserving liquid C 2, E 2the holding time that sperm viability reaches 80% is respectively 156h and 144h.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.

Claims (2)

1. the nearly red Culter sperm storage method of black tail, it is characterized in that, concrete steps are as follows:
(1) preparation of spermatozoa preservative fluid: take each material composition by proportioning, dissolve respectively with distilled water, mix, adding distil water is settled to 100mL;
Spermatozoa preservative fluid adopts the one in following formula to prepare:
A 1conserving liquid is filled a prescription: NaCl0.80g, KCl0.04g, CaCl 20.01g, MgSO 47H 2o0.02g, NaHCO 30.04g, Na 2hPO 4h 2o0.02g, KH 2pO 40.01g, C 6h 12o 6h 2o0.10g, distilled water is settled to 100mL;
A 2conserving liquid is filled a prescription: NaCl0.80g, KCl0.04g, CaCl 20.01g, MgSO 47H 2o0.02g, NaHCO 30.04g, Na 2hPO 4h 2o0.02g, KH 2pO 40.01g, C 6h 12o 6h 2o0.10g, penicillin 200 × 10 4iU, distilled water are settled to 100mL;
B 1conserving liquid is filled a prescription: NaCl0.7800g, KCl0.0200g, CaCl 20.0210g, NaHCO 30.0021g, distilled water are settled to 100mL;
B 2conserving liquid is filled a prescription: NaCl0.7800g, KCl0.0200g, CaCl 20.0210g, NaHCO 30.0021g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
C 1conserving liquid is filled a prescription: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, distilled water is settled to 100mL;
C 2conserving liquid is filled a prescription: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
D 1conserving liquid is filled a prescription: NaCl0.743g, KCl0.278g, CaCl 20.029g, MgCl 26H 2o0.030g, NaHCO 30.042g, distilled water is settled to 100mL;
D 2conserving liquid is filled a prescription: NaCl0.743g, KCl0.278g, CaCl 20.029g, MgCl 26H 2o0.030g, NaHCO 30.042g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
E 1conserving liquid is filled a prescription: NaCl0.6903g, KCl0.0946g, CaCl 20.0300g, MgCl 26H 2o0.0142g, Na 2hPO 40.0540g, NaHCO 30.3999g, TYR 0.0996g, glycine 0.0412g, bovine serum albumin(BSA) 0.4110g, distilled water is settled to 100mL;
E 2conserving liquid is filled a prescription: NaCl0.6903g, KCl0.0946g, CaCl 20.0300g, MgCl 26H 2o0.0142g, Na 2hPO 40.0540g, NaHCO 30.3999g, TYR 0.0996g, glycine 0.0412g, bovine serum albumin(BSA) 0.4110g, penicillin 200 × 10 4iU, distilled water is settled to 100mL;
(2) semen collection: towel off the nearly red Culter milter of dry ripe black tail and get rid of urine and ight soil, avoiding seminal fluid to pollute; Light pressure belly, collect the seminal fluid extruded with suction pipe, gatherer process avoids sunlight or Ultraviolet radiation;
(3) seminal fluid is deposited: be placed in by near for the black tail of collection red Culter seminal fluid through the drying of sterilization, cleaning and the container of sealing, the volume ratio of seminal fluid and spermatozoa preservative fluid is 1:5 ~ 10, gathers after terminating and the container filling seminal fluid and conserving liquid is put 3 ~ 5 DEG C of low-temperature darks preservations.
2. the nearly red Culter sperm storage method of black tail according to claim 1, is characterized in that, in described step (1), the preferred version of the nearly red Culter spermatozoa preservative fluid of black tail is conserving liquid formula C 2: glucose 2.90g, KCl0.05g, CaCl 20.02g, NaHCO 30.21g, penicillin 200 × 10 4iU, distilled water is settled to 100mL, under 4 DEG C of conditions, conserving liquid formula C 2the holding time making the nearly red Culter sperm viability of black tail reach 80% is 156h.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107047540A (en) * 2017-03-29 2017-08-18 华南农业大学 A kind of Chang Tun Catfish seminal fluid cryopreservation method and its dilution
CN109874707A (en) * 2019-04-08 2019-06-14 中国科学院水生生物研究所 A kind of method of efficient initiative allooctaploid pond crucian carp

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CN104304236A (en) * 2014-09-30 2015-01-28 采克俊 Erythroculter ilishaeformis semen cryopreservation solution

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EP1223805B1 (en) * 1999-10-20 2004-02-18 Istituto Sperimentale Italiano " Lazzaro Spallanzani" Method for cryopreservation of teleostei semen
CN104304236A (en) * 2014-09-30 2015-01-28 采克俊 Erythroculter ilishaeformis semen cryopreservation solution

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107047540A (en) * 2017-03-29 2017-08-18 华南农业大学 A kind of Chang Tun Catfish seminal fluid cryopreservation method and its dilution
CN107047540B (en) * 2017-03-29 2020-12-29 华南农业大学 Ultralow-temperature preservation method for seminal fluid of leiocassis longirostris and diluent thereof
CN109874707A (en) * 2019-04-08 2019-06-14 中国科学院水生生物研究所 A kind of method of efficient initiative allooctaploid pond crucian carp

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