92 results about "Sperm cryopreservation" patented technology

A practical method for freeze storage of fish sperm features that the diluent, antifreezing agent and container for the freeze storage of fish sperm chosen from many schemes, and a three-step freezing mode and a quick thawing method are designed.

The invention relates to a method for freezing and storing diluted semen of cow. Wherein, the invention is characterized in that: preparing said diluted semen into A, B, C, D deals; mixing them with right ratio; adding 5-20% pig semen into C and D deals, to improve the semen acrosome integral rate of cow semen control liquid, and reduce the density of semen, to save cost. The experiment has proved that the semen survival rate of stored diluted semen can reach 50.88-62.88%, while the average number is 57.41%, which is 15.13% higher than traditional method, and the semen acrosome integral rate can reach 50.88-53.20%, while the average number is 51.90%, which is 7.0% higher than traditional method.

The invention discloses a method for inducing cleavage gynogenesis of fish fry of cynoglossus semilaevis, which comprises the steps of cynoglossus semilaevis sperm freezing storage, ultraviolet radiation of sperms, and induction and identification of the cleavage gynogenesis of the fish fry. The cynoglossus semilaevis sperm freezing storage method is established, and a method for inhibiting the first cleavage to induce the multiplication of embryo chromosome of the gynogenesis of the cynoglossus semilaevis through hydrostatic pressure is adopted so as to screen effective dilution and freezing method for the cynoglossus semilaevis sperm freezing storage and screen the hydrostatic pressure shock starting time, hydrostatic pressure and processing time suitable for the cleavage gynogenesis of cynoglossus semilaevis; and an identification method for the gynogenesis of the fish fry of the cynoglossus semilaevis is established. In the method, the fish fry of the cynoglossus semilaevis through cleavage gynogenesis is obtained for the first time, and the inductivity of the gynogenesis of the fish fry reaches 0.25 percent; and the method has the characteristics of advancement, high efficiency, reliability and reliability, can be used for solving the problems that male cynoglossus semilaevis grows slowly, the ratio of female cynoglossus semilaevis is low and the culture cost is high, and has significant application value and wide promotion and application prospect in the aspects of breeding of cynoglossus semilaevis fry, sex control, culture of all female fry, pure line breeding and the like.

The invention discloses a trace sperm cryopreservation carrier and corresponding freezing and thawing methods thereof. The trace sperm cryopreservation carrier is in a rectangular, square or circular horizontal or groove shape and is made of a transparent plastic or transparent glass, and the size of the trace sperm cryopreservation carrier can bear 1-3 0.1-10.0 mu L freezing liquid micro drops. The freezing and thawing method comprises the following steps: after preparing the freezing liquid micro drops on the trace sperm cryopreservation carrier, inserting the trace sperm cryopreservation carrier into a sperm culture dish covered with oil, grabbing sperms by a microinjection needle, putting into the freezing liquid micro drops, taking out the cryopreservation carrier, soaking up mineral oil by virtue of an absorbent paper, rapidly freezing, directly inserting the cryopreservation carrier into an ICSI vessel when thawing, and thawing the sperms by utilizing the mineral oil with the temperature of 37 DEG C. According to the trace sperm cryopreservation carrier and the corresponding freezing and thawing method thereof disclosed by the invention, as the cryopreservation carrier is inserted into the sperm culture dish covered with oil, and the sperms are grabbed and frozen, a special moisture retention device is not needed, and evaporation of the freezing liquid micro drops is not needed to be worried about; the operation is very simple, the flexibility and reliability of assisted reproductive treatment for a patient with azoospermia are greatly improved, the sperm recovery rate can reach 100 percent, and the sperm viability is about 60 percent.

The invention discloses acipenser dabryanus sperm preserving fluid liquid and a preparing method and application. The acipenser dabryanus sperm preserving fluid liquid comprises saccharose, mycose, trihydroxy aminomethane, potassium chloride, reduced glutathione and carbinol. The acipenser dabryanus sperm preserving fluid liquid has simple components and is low in cryopreservation cost. The added reduced glutathione plays a good role in antioxygenation, well protects sperm plasma membranes and reduces damage to DNA molecules. The added mycose has the effects of preventing protein denaturation and effectively protecting sperms. By means of the acipenser dabryanus sperm preserving fluid liquid, motility of unfreezed sperms is higher than 70%, and the fertility rate reaches 45%.

A bastard halibut sperm rapid cryopreservation method relates to a cryopreservation method for marine fish sperm and provides a bastard halibut sperm cryopreservation method with simple operation, rapid preservation and high sperm living rate. The present invention contains diluent, antifreeze agent and the confection of hormone cryoprotective liquid and is preserved in liquid nitrogen. The present invention is characterized in that the diluted natural seawater is used as the diluent for preserving sperms which can not activate sperms; glycol with 14g/100ml density is adopted as the anti-freeze agent; the diluent and the antifreeze agent are used as the low-temperature hormone cryoprotective liquid for bastard halibut sperms and the low-temperature hormone cryoprotective liquid is precooled in a 4 DEG C thermostatic box; the sperm and the hormone cryoprotective liquid are evenly mixed in an eccentric pipe according to the proportion of 1:1 to 1:3; the eccentric pipe is placed below the port of a liquid nitrogen jar for balancing under -20 DEG C for 1 minutes and then rapidly thrown into the liquid nitrogen for preservation. The present invention is used for bastard halibut sperm rapid cryopreservation.

The invention discloses a sperm cryopreservation method of Charybdisjaponica, which is characterized by comprising the steps of material drawing, sperm activity identification, cooling balance, first-time precooling, second-time precooling, freezing and preserving and the like. Aiming at the biological characteristics of Charybdisjaponica sperm, the invention screens out a cryopreservation method suitable for Charybdisjaponica sperm, which can effectively preserve Charybdisjaponica sperm for a long term. After the Charybdisjaponica sperm is preserved in liquid nitrogen for one year by utilizing the method of the invention, the livability of unfrozen sperm can reach over 70%. The method has no need of expensive apparatus, simple operation, low cost and good effect, provides source guarantee of sperm for the artificial insemination of Charybdisjaponica, and can be used for other research fields such as hybridization breeding, germplasm preference, gynogenesis, species conservation and the like of Charybdisjaponica.

The invention discloses an efficient grouper sperm cryopreservation solution and a use method thereof, and belongs to the technical field of fish germplasm cryopreservation. The efficient grouper sperm cryopreservation solution is prepared from diluent and an antifreeze agent; the diluent is an aqueous solution and is prepared from, by mass, 10-20 g/L of glucose, 7.5-8.5 g/L of sodium chloride and 0.6-0.7 g/L of potassium chloride; the antifreeze agent is 1,2-propylene glycol, and the volume concentration of the antifreeze agent in the cryopreservation solution is 10-20%. The use method of the cryopreservation solution for preserving grouper sperms comprises the seven steps of diluent preparation, antifreeze agent preparation, cryopreservation solution precooling, sperm dilution, balancing and subpackaging, cryopreservation, unfreezing and fertilization. The cryopreservation solution is simple in component, low in toxicity, easy to prepare, easy to master by breeding technicians and high in freezing activity, high universality is achieved, and the grouper sperm cryopreservation survival rate is increased.

The invention provides cryopreservation solution of horse sperms and a freezing method thereof. The freezing method comprises the steps of firstly, applying mare milk and yolk low-density lipoprotein after special treatment to the cryopreservation solution of horse sperms at the same time; adding the cryopreservation solution by sub-steps in the freezing process; and controlling cooling, lower liquid nitrogen fumigation height and shorter fumigation time. The quality of freezing the horse sperms is obviously improved by the technology; the artificial insemination efficiency of frozen semen of the horse is greatly improved.

The invention discloses an application of a polysaccharide substance in preparation of a dilution solution for freeze preservation of goat sperms and a preparation method of the dilution solution. Thepolysaccharide substance comprises one or more selected from the group consisting of tremella polysaccharide, lentinan, auricularia polysaccharide and cordyceps polysaccharide. The invention also provides the dilution solution for freeze preservation of the goat sperms. The dilution solution comprises the polysaccharide substance, a nutrient substance, a buffer substance and the like. The dilution solution for freeze preservation of the goat sperms provided by the invention has the effects of improving a sperm motility rate, an acrosome and plasma membrane integrity rate after freeze preservation, and significantly improving goat reproductive performance and economic benefits.

The invention discloses a method for establishing a giant grouper sperm freezer and assisting grouper distant hybridization breeding. The sperm freezer establishing method comprises the steps of preparing sperm diluent, preparing sperm cryopreservation liquid, freezing and storing sperms and establishing the sperm freezer. The grouper distant hybridization breeding method comprises the steps of establishing the giant grouper sperm freezer, establishing saladfish breeding groups, cultivating saladfish parent fish and performing reproductive regulation, performing artificially spawning breeding, fertilizing the frozen sperms and performing incubation, and cultivating fries. According to the method, the first giant grouper sperm freezer is established in China, reproductive isolation of the groupers caused by factors, such as geographical distribution, optimum temperature range and breeding time, is overcome, and distant hybridization breeding of the grouper groups is possible, so that distant hybridization breeding varieties are cultivated.

The present invention relates to a method for collecting high-concentration seminal fluid of scallop. Said method includes the following several procedures; incubation of high-quality parent scallop, preparation of suction head for sucking seminal fluid, collection of high-concentration seminal fluid and detecting vitality of collected spermatozoa. Said invention also provides the concrete requirements and steps of above-mentioned every procedure.

The invention relates to a pomfret sperm cryopreservation method including the steps as follows: selecting sexually matured parental pomfrets to gather fine-quality sperm; selecting suitable base liquid and anti-freezing liquid; mixing the sperm with the anti-freezing liquid by the volume ratio of 1:1 and then preserving the mixture on ice for 4-5 min; after reducing the temperature to minus 150 DEG C according to a three-step temperature reduction method, staying for 5 min; directly placing the mixture in liquid nitrogen for preservation; in defreezing, staying a freeze pipe in liquid nitrogen steam for 2 min, rapidly placing the freeze pipe in water bath of 28 DEG C to defreeze for 90 s and then placing the freeze pipe at room temperature to dissolve completely; and activating the defrozen sperm with sea water, wherein the rapid swimming time of the sperm is 57.41 s averagely, and the sperm viability (percent of the sperms moving rapidly accounting for all sperms) is 83.62% averagely. The fertilization rate of the pomfrets is greatly enhanced.

The present invention relates to method of increasing sperm and freeze preserving sperm for fish, and is especially the method of increasing sperm and freeze preserving sperm of verasper variegates as marine fish. The method includes selecting male verasper variegates capable of extruding out milk white sperm, injecting HCG in the amount of 150 IU/kg to abdominal cavity to increase sperm, adding antifreeze fluid to the obtained sperm and pre-cooling at 0-4 deg c for 9-11 min, sectionally lowering the temperature to ¿C180 deg c and maintaining in liquid nitrogen. During defrosting, the frozen sperm is first set inside warm water at 43 deg c to slight thawing and then defrosted at room temperature to obtain sperm with fertilizing capacity. The present invention is significant in breeding verasper variegates as one kind of rare marine fishes, germ plasm maintenance, genetic diversity and other aspects.

The invention discloses an acipenser schrenckii cryopreservation fluid and a long-term acipenser schrenckii sperm cryopreservation method. The pH of each liter of acipenser schrenckii cryopreservationfluid is 7.5-8.5. The acipenser schrenckii cryopreservation fluid is prepared from 20-30 mM of sucrose, 0.1-0.5 mM of potassium chloride, 20-40 mM of Tris-HCl, 5-10 g/L of bovine serum albumin, 2.5-10mM of reduced glutathione and antifreeze components and the balance ultra-pure water, wherein the antifreeze components are, by volume, one of 10% of methanol, 15% of DMSO, the mixture of 15% of DMSOand 10% of fresh egg yolk, 15% of propylene glycol and the mixture of 15% of propylene glycol and 10% fresh egg yolk mixture. The components of the acipenser schrenckii cryopreservation fluid are clear and definite, can protect sperms against damage caused by ice crystal, oxidization and the like, the fertilization rate can reach 45.87% after cryopreserved sperms are unfrozen and revive, healthyfry can be hatched, and the acipenser schrenckii cryopreservation fluid has the advantages of being simple, convenient, easy to operate and capable of completing cryopreservation without precious instruments when being applied to long-term acipenser schrenckii sperm cryopreservation.

The invention relates to a preparation method of a soya lecithin frozen diluent. Firstly, TCG liquid is prepared, soya lecithin is added to the preheated prepared TCG liquid in a weight percentage of1-10%, and the soya lecithin and the TCG liquid are uniformly stirred and then subjected to ultrasonic treatment to obtain a mixed solution; and finally, the mixed solution is subjected to high-pressure homogenization treatment to obtain the clear and transparent soya lecithin frozen diluent with stable properties and no precipitation after being placed for a long time. The soya lecithin frozen diluent prepared by the preparation method of the invention is used for cryopreservation of sheep semen after adding glycerol, which can improve the solubility of the soya lecithin and increase the stability of the solution by greatly reducing the particle size of the soya lecithin in the frozen diluent and has good effect on sperm cryopreservation.

The invention relates to urechis unicinctus sperm cryopreservation liquid which comprises urechis unicinctus sperms, preservatives and antifreeze agents. A preparation method of the cryopreservation liquid includes the steps: collecting sperms; identifying sperm activity; diluting the sperms; reducing temperature, and balancing; firstly pre-cooling; secondly pre-cooling; preserving; unfreezing; eluting and the like. According to the cryopreservation liquid, the urechis unicinctus sperms can be effectively preserved for a long time and are preserved in liquid nitrogen, and activation rate of unfrozen sperms can reach 70% or more. According to the preparation method, expensive instruments are omitted, and the preparation method is simple to operate, low in cost and good in effect, provides sperm source guarantee for artificial insemination operations of urechis unicinctus and can be used for other research fields such as crossbreeding, germplasm selection, gynogenesis and species protection of the urechis unicinctus.

The invention discloses a human testicular tissue suspension cryoprotectant and a preparation method thereof. The human testicular tissue suspension cryoprotectant comprises the following raw materials: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, monopotassium phosphate, sodium bicarbonate, HEPES, caffeine, sodium pyruvate, pentoxifylline, glycine, glucose and sucrose. The human testicular tissue suspension cryoprotectant also comprises sodium lactate and glycerol. The invention further discloses the preparation method of the human testicular tissue suspension cryoprotectant and a freezing method adopting the human testicular tissue suspension cryoprotectant. The cryoprotectant is specially designed for a testicular tissue suspension, the testicular sperm cryopreservation and resuscitation effect can be improved, the problems that potential safety hazards exist, the cryopreservation and resuscitation effect is poor, and movable testicular sperms are not easy to find are effectively solved, and the human testicular tissue suspension cryoprotectant has quite important clinical application value.

The invention discloses a rat sperm cryopreservation agent (RS-CPA) for a SD rat and a gene engineering rat under a SD rat genetic background. The RS-CPA comprises 5% of beam milk extract as a RS-CPA main component and also comprises raffinose. Through use of the beam milk extract, the RS-CPA greatly improves resurgent sperm vitality and a fertilization rate and realizes a refrigeration recovery rate of 10% or more.

The invention belongs to the technical field of marine organisms, particularly a method for carrying out interspecies cross artificial insemination on an egg of paralichthys olivaceus and a semen of paralichthys dentatus, which is frozen for preservation. The semen of paralichthys dentatus, which is frozen for preservation at an ultralow temperature for a long time, is dissolved in water by two steps; after the semen of paralichthys dentatus is dissolved, diluent of which the volume is three times of that of the frozen semen of paralichthys dentatus is added into the frozen semen of paralichthys dentatus; after the frozen semen of paralichthys dentatus and the diluent are mixed, centrifugation is carried out for 5min; then supernate is removed to obtain frozen semen suspension with the same volume with the frozen semen of paralichthys dentatus before the frozen semen of paralichthys dentatus is diluted; the frozen semen and natural seawater are uniformly mixed according to the proportion of a volume ratio of 1:40 and the mixture is activated; after the mixture is activated, the egg of paralichthys olivaceus is injected and the mixture and the egg of paralichthys olivaceus are slightly and uniformly mixed to enable the egg of paralichthys olivaceus and the frozen semen of paralichthys dentatus to be sufficiently in contact; standing is carried out for 5 to 10min and egg washing is carried out to obtain a fertilized egg to carry out incubation. The invention establishes a cryopreservation technology for semen of paralichthys dentatus and a method for interspecies cross artificial insemination on the egg of paralichthys olivaceus, overcomes the difficulty of asynchrony of development of parental fishes of paralichthys dentatus and paralichthys olivaceus and provides a novel method and a novel idea for seawater fish cross breeding.