CN114403131A - Semen cryopreservation liquid and application thereof - Google Patents
Semen cryopreservation liquid and application thereof Download PDFInfo
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- CN114403131A CN114403131A CN202210071955.0A CN202210071955A CN114403131A CN 114403131 A CN114403131 A CN 114403131A CN 202210071955 A CN202210071955 A CN 202210071955A CN 114403131 A CN114403131 A CN 114403131A
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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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- 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
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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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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Abstract
The invention discloses a semen cryopreservation liquid and application thereof, and belongs to the technical field of cryopreservation. The semen cryopreservation liquid is prepared from Tris, citric acid, yolk, ethylene glycol, vitamin C, bioflavonoids and trehalose according to a specific ratio, so that the vitality and the survival rate of frozen and revived sperms can be maintained, the oxidative stress reaction caused by freezing is reduced, the acrosome and the membrane structure of the sperms are damaged, and a foundation is laid for a high-vitality sperm freezing method.
Description
Technical Field
The invention belongs to the technical field of frozen storage, and particularly relates to semen frozen preservation solution and application thereof.
Background
The silver black fox is a seasonal single-estrus animal, the estrus is concentrated from the middle and last ten days of 1 month to the middle and last 3 months of each year, the estrus duration is 5-10 days, the silver black fox can continuously mate for 2-4 times in the whole estrus, the estrus period is as long as 10 months, the breeding efficiency is extremely low, and the development of the fox industry is seriously influenced.
At present, the normal-temperature semen diluent and the artificial insemination technology of the silver black fox are mature, but the research on the diluent for freezing and storing the semen of the silver black fox at home and abroad is little, and the normal-temperature semen diluent and the artificial insemination technology of the silver black fox are not applied and popularized in a large scale. The frozen semen artificial insemination is carried out, so that the mating potential of the male foxes of the superior variety can be improved, the semen of the male foxes of the superior variety can be effectively preserved, the breeding process is accelerated, the feeding quantity of the male foxes in the actual production can be reduced, the feeding cost is saved, and the condition that a large number of male foxes cannot be used in the later stage of mating is relieved.
The existing diluent for freezing and preserving the semen of the black silver fox has a plurality of problems when used for freezing and preserving the semen and artificial insemination, and mainly comprises the following steps: firstly, sperms are difficult to resist damage caused by rapid freezing of liquid nitrogen, so that a large number of sperms die; secondly, the freezing easily causes oxidative stress reaction to destroy the sperm acrosome and membrane structure; the sperm activity is greatly reduced after thawing and reviving, the in vitro survival time is short, the reproduction rate is very low after artificial insemination, and the semen waste of the male fox of excellent quality is caused; the current reports mostly focus on the research of fast liquid nitrogen freezing after the fresh semen of the silver black fox is collected, then the fresh semen is unfrozen and inseminated in the same day, and the semen quality identification and the artificial inseminating results evaluation lack of long freezing preservation time.
Therefore, how to provide the fox semen cryopreservation liquid and the application thereof are problems to be solved in the field.
Disclosure of Invention
The invention discloses a fox semen cryopreservation liquid and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a semen cryopreservation liquid comprises: tris 0.12g/L, citric acid 0.08 g/L, yolk 2ml/L, ethylene glycol 0.6ml/L, vitamin C6 g/L, bioflavonoid 0.8g/L and trehalose 40-90 mmol/L;
preferably, the working concentration is 2/3 of the original concentration;
the original concentration is the concentration of undiluted working concentration;
preferably, the method further comprises the following steps: gentamicin with the original concentration of 100 ten thousand UI/L;
the application of the semen cryopreservation liquid in the preparation of a semen storage preparation;
the application of the semen cryopreservation liquid in preparing a fox semen storage preparation;
a semen cooling cryopreservation method comprises the following steps:
semen collection:
collecting fox semen at a frequency of 2-3 days with interval of 1-2 days;
freezing semen:
mixing semen cryopreservation solution with nutria semen at working concentration to obtain pre-refrigerated semen, and pre-refrigerating the pre-refrigerated semen at 4 deg.C; then precooling to below-10 ℃ by using liquid nitrogen steam, and then quickly putting into liquid nitrogen for preservation;
preferably, precooling is to put the mixture into a refrigerator at 4 ℃ until the mixture is completely precooled;
preferably, the mixture is placed for 2 hours;
preferably, liquid nitrogen steam is precooled to the temperature below-20 ℃;
preferably, liquid nitrogen steam is precooled to below-30 ℃;
a semen cooling unfreezing method comprises the following steps:
unfreezing:
taking out the frozen semen according to claim 4, standing at room temperature for 10min, transferring to 70 deg.C environment until the semen is completely dissolved, taking out, mixing semen and thawing solution at 1:2-1:1, and thawing in 22-24 deg.C thawing solution;
preferably, the semen and the unfreezing liquid are mixed uniformly according to the ratio of 1: 2;
preferably, 0.5ml of semen is taken;
the semen is fox semen;
preferably, the formula of the thawing solution is as follows: 100ml of distilled water, 1.7g of sodium citrate, 1.15g of cane sugar, 0.09g of sodium bicarbonate, 0.35g of monopotassium phosphate, 0.3g of sulfanilamide and 5-10 ten thousand units of streptomycin respectively;
in conclusion, the invention discloses a semen cryopreservation liquid and application thereof, the semen cryopreservation liquid is prepared from Tris, citric acid, egg yolk, ethylene glycol, vitamin C, bioflavonoid and trehalose according to a specific ratio, the vitality and the survival rate of frozen and revived sperms can be maintained, the oxidative stress reaction caused by freezing is reduced to damage the acrosome and membrane structure of the sperms, and a foundation is laid for a high-vitality freezing method of the sperms.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Materials and methods
Primary reagents and instruments
The main components used for preparing the diluent are as follows: tris (hydroxymethyl) aminomethane (Tris), citric acid, egg, ethylene glycol, penicillin, streptomycin, trehalose, vitamin C, bioflavonoids, potassium dihydrogen phosphate, and disodium hydrogen phosphate. Sperm vital staining fluid (eosin-nigrosine method), coomassie brilliant blue kit.
The instruments and consumables used include: a fox retaining frame, an electric heating constant temperature water bath, a liquid nitrogen tank, a microscope (DM6B, Germany LEICA), an ultra-clean bench, an acidimeter, a centrifuge, a semen collection cup, a full-automatic sperm analyzer (TOX IVOS, Hamilton Thorne, USA), a cell counting plate, a fox stainless steel sperm feeder, a fox sperm feeding glass sleeve and a frozen sperm tubule.
Laboratory animal
The formula of the thawing solution comprises:
100ml of distilled water, 1.7g of sodium citrate, 1.15g of cane sugar, 0.09g of sodium bicarbonate, 0.35g of monopotassium phosphate, 0.3g of sulfanilamide and 5-10 ten thousand units of streptomycin respectively.
All experimental animals were bred in the city of Matou Yingzhen, Lepavilion county, Tangshan, Hebei province. 5 healthy male foxes of black and silver foxes in the mating period are selected, fed for 1 time respectively at 08:00 am and 15:00 pm every day, and fed freely and drunk freely.
Preparation of freezing preservation diluent
Weighing 1.2g of Tris, 0.8g of citric acid, 20ml of yolk, 6ml of ethylene glycol, 10 ten thousand UI/100ml of gentamicin, 6mg/ml of vitamin C, 0.8mg/ml of bioflavonoid, trehalose (added with 6 different concentrations, namely 40mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L and 90mmol/L respectively), and fixing the volume to 100ml by double distilled water.
Semen collection
Collecting semen of fox in clean semen collecting cup (placed in 37 deg.C water bath) with scale by hand massage. Semen is collected for 2-3 times per week, and the male fox should rest for 1-2 days for 2-3 consecutive days, and the number of semen collection times cannot be increased at will to prevent the quality of semen from being reduced, which leads to the adverse effects of reducing the utilization rate of male foxes, etc.
Preparation and unfreezing of frozen semen of thin tube
Before freezing, diluting semen according to the proportion of 1:2, balancing the diluted semen in a refrigerator at 4 ℃ for 2h, subpackaging the balanced semen by using a frozen semen tubule, and sealing by using a sealing machine. Then horizontally placing the tubule on a prepared tubule bracket (5 cm from the liquid nitrogen horizontal plane), covering the cover of the freezing tank, fumigating for 10s on the liquid nitrogen surface, and quickly filling the tubule into liquid nitrogen for storage. Taking a 500mL big beaker during thawing, pouring warm water at 70 ℃, taking out a thin tube stored in liquid nitrogen, standing for 10min at room temperature, quickly placing in the warm water to slowly stir, quickly taking out the thin tube when the semen is completely dissolved, cutting two ends, adding the semen into 0.5mL of unfreezing liquid at 22-24 ℃, placing the unfreezing liquid containing the semen into a CO2 incubator, and culturing at 37 ℃ for subsequent semen quality detection.
Semen quality detection
Firstly, sperm motility detection: the main parameters of semen quality were determined with a fully automated sperm analyzer (Waite et al, 2008). The parameters of semen quality include: sperm density (. times.10)6sperm/mL), motile sperm ratio (%), pathway velocity (VAP, μm/s), linear motion (VSL, μm/s), curvilinear motion (VCL, μm/s), and preformance (STR [ VAP/VCL ])]100, respectively; %), linearity (LIN ═ VSL/VCL)]100；％)。
Secondly, detecting the integrity of the plasma membrane: taking 1ml of hypotonic solution, preheating in a water bath kettle at 37 ℃ for min, adding the diluted semen into the preheated hypotonic solution, incubating for 30min, counting 200 sperms under a high power microscope, and calculating the percentage of the tail of the sperms with swelling.
Checking the integrity of the acrosome (Coomassie brilliant blue staining method): adding 80ul semen diluentCentrifuging in lml 0.9% physiological saline at 10000 rpm for 15s, and removing the supernatant; adding 1ml of 3.7% paraformaldehyde/PBS, suspending, fixing at room temperature for 30min, centrifuging and removing supernatant; suspending with lml PBS, centrifuging and discarding the supernatant; suspending with 500-800u1 PBS and drying the smear; staining with 0.22% Coomassie Brilliant blue (prepared with 50% methanol and 10% glacial acetic acid in water) for 5 min; by dH2Wash off stain and dry, seal with neutral gum, observe 200 sperm, acrosome blue, and sperm with smooth acrosome edges are of the type with intact acrosomes.
Fresh semen analysis
After semen collection, the fox semen diluent (Xinyuan, California) is used for diluting the semen according to the proportion of 1:2, and the semen is rapidly subjected to routine quality inspection. The results showed that 5 fox sperm concentration, motile sperm ratio, pathway Velocity (VAP), linear motion (VSL), curvilinear motion (VCL), forward (STR), Linearity (LIN), plasma membrane integrity and acrosome integrity differences were not significant (P > 0.05) (table 1).
TABLE 1 quality determination results of fresh semen of different individual silver black foxes
Influence of trehalose addition at different concentrations on semen quality after freezing-thawing
As can be seen from Table 2, the difference between the sperm concentration and LIN was not significant between the 6 groups (P > 0.05); the ratio of active sperm is 50mmol/L, the ratio of 60mmol/L addition group is obviously higher than 40mmol/L, the ratio of 80mmol/L and 90mmol/L addition group (P <0.05), the difference with 70mmol/L addition group is not significant (P > 0.05), and the ratio of 70mmol/L addition group is obviously higher than 40mmol/L and 90mmol/L addition group (P < 0.05); the VAP60mmol/L addition group is obviously higher than 40mmol/L, 70mmol/L, 80mmol/L and 90mmol/L addition group (P <0.05), and the difference with the 50mmol/L addition group is not significant (P > 0.05); the VSL60mmol/L addition group is obviously higher than 40mmol/L, 50mmol/L, 80mmol/L and 90mmol/L addition group (P <0.05), and the difference with the 70mmol/L addition group is not significant (P > 0.05); the VCL50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L addition groups did not differ significantly (P > 0.05), but were significantly higher than the 40mmol/L and 90mmol/L addition groups (P < 0.05); STR and plasma membrane integrity rates of 50mmol/L, 60mmol/L, 70mmol/L were not significantly different in the addition groups (P > 0.05), but were significantly higher than in the 40mmol/L, 80mmol/L, and 90mmol/L addition groups (P < 0.05); the acrosome integrity rate of 60mmol/L addition group is obviously higher than that of 40mmol/L, 50mmol/L, 70mmol/L, 80mmol/L and 90mmol/L addition group (P < 0.05).
Comparative example 1
In the actual production, as the formula of the silver-black fox cryopreservation liquid is not adopted, the blue fox cryopreservation liquid is adopted to dilute the silver-black fox semen for cryopreservation.
The existing blue fox cryopreservation liquid formula comprises the following components:
20ml of yolk, 4ml of glycerol, 3.8g of fructose and 1.2IU of penicillin; as a control group i.
240mmol/L Tris, 63mmol/L sodium citrate, 20ml yolk, 4ml glycerin and 70mmol/L trehalose; as a control group ii. The rest of the operation was the same as in example 1, and the results are shown in Table 2.
As can be seen from table 2, LIN was not significantly different between 6 experimental groups and 2 control groups (P > 0.05); sperm concentration and VSL and STR for control group i were significantly lower than 50mmol/L and 60mmol/L addition groups (P <0.05), not significantly different from the other 4 experimental groups (P > 0.05), motile sperm ratio and plasma membrane integrity rate significantly lower than 50mmol/L, 60mmol/L and 70mmol/L addition groups (P <0.05), not significantly different from the other 3 experimental groups (P > 0.05), VAP, VCL and STR not significantly different from the 90mmol/L addition group (P > 0.05), but significantly lower than the other 5 experimental groups (P <0.05), plasma membrane integrity rate not significantly different from the 40mmol/L and 90mmol/L addition groups (P > 0.05), but significantly lower than the other 4 experimental groups (P < 0.05); the ratio of motile sperm, VAP, VCL, plasma membrane integrity and acrosome integrity in control group II were significantly lower than those in 6 experimental groups (P <0.05), and the sperm concentration, VSL and STR in control group II were not significantly different from those in 90mmol/L addition group (P > 0.05), but significantly lower than those in the other 5 experimental groups (P < 0.05).
TABLE 2 Effect of trehalose addition at different concentrations on semen quality after freezing-thawing
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A semen cryopreservation liquid is characterized by comprising: tris 0.12g/L, citric acid 0.08g, L, yolk 2ml/L, ethylene glycol 0.6ml/L, vitamin C6 g/L, bioflavonoid 0.8g/L and trehalose 40-90 mmol/L.
2. The semen cryopreservation liquid as claimed in claim 1, wherein the working concentration is 2/3 of the original concentration.
3. The semen cryopreservation liquid as claimed in claim 1 or 2, further comprising: gentamicin with the original concentration of 100 ten thousand UI/L.
4. Use of the semen cryopreservation solution as claimed in any one of claims 1 to 3 in the preparation of a semen storage preparation.
5. Use of a semen cryopreservation solution as claimed in any one of claims 1 to 3 in the preparation of a fox semen storage formulation.
6. A semen cooling cryopreservation method is characterized by comprising the following steps:
semen collection:
collecting fox semen at a frequency of 2-3 days with interval of 1-2 days;
freezing semen:
mixing semen cryopreservation solution with nutria semen at working concentration to obtain pre-refrigerated semen, and pre-refrigerating the pre-refrigerated semen at 4 deg.C; then precooling to below-10 ℃ by using liquid nitrogen steam, and then quickly storing in liquid nitrogen.
7. The thawing method of the semen cooling is characterized by comprising the following steps:
unfreezing:
taking out the frozen semen according to claim 4, standing at room temperature for 10min, transferring to 70 deg.C environment until the semen is completely dissolved, taking out, mixing semen and thawing solution at 1:2-1:1, and thawing in 22-24 deg.C thawing solution.
8. The semen thawing method of claim 7, wherein the formulation of the thawing solution is: 100ml of distilled water, 1.7g of sodium citrate, 1.15g of cane sugar, 0.09g of sodium bicarbonate, 0.35g of monopotassium phosphate, 0.3g of sulfanilamide and 5-10 ten thousand units of streptomycin respectively.
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