CN101616578A - The preservation of sperm and controlled sending are passed/are discharged - Google Patents
The preservation of sperm and controlled sending are passed/are discharged Download PDFInfo
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- CN101616578A CN101616578A CN200780025213A CN200780025213A CN101616578A CN 101616578 A CN101616578 A CN 101616578A CN 200780025213 A CN200780025213 A CN 200780025213A CN 200780025213 A CN200780025213 A CN 200780025213A CN 101616578 A CN101616578 A CN 101616578A
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Abstract
The present invention relates to be used for the biopolymerization composition granule of preservation sperm, wherein said sperm is embedded in the described biopolymerization composition granule.The method that the present invention also passs/discharges about a kind of preservation, storage and controlled sending of sperm, and the purposes of biopolymerization composition granule of the present invention in breeding.
Description
Technical field
The present invention relates to be used for the biopolymerization composition granule of preservation sperm.The invention still further relates to the preservation that is used for sperm, storage and controlledly send the method for passing/discharging, and the purposes of biopolymerization composition granule of the present invention in breeding.
Background technology
Artificial insemination (AI) is a kind of such technology, promptly wherein by manual type rather than sperm is placed uterus or the uterine neck of animal by natural mating.It is widely used as a kind of spouse's method, and is used to animal breeding to breed required proterties, particularly for farm-animals for example ox, pig, sheep, poultry and horse, and for pet, aquatic animal or the endangered species of for example purebred dog.
Usually, sperm is gathered, is diluted, then by for example cryopreservation preservation.The cryopreservation The Application of Technology presupposes: the sperm from the particular animals kind tolerates this processing, and does not cause the excessive deterioration of sperm quality, vigor and fertility.Then the described sperm (which kind of all is suitable) of cryopreservation or fresh storage is delivered to the position of described jenny.Any is arranged is vital, and promptly described sperm still maintains vigour when insemination, and this vigor keeps the sufficiently long time to arrive the described site of fertilization until egg cell in the jenny body after the insemination.
The artificial insemination of farm-animals was just brought into use from the forties in 20th century, and was widely used in now in the agricultural industry, in particular for breeding milk cow and pig.Modern AI development and breeder are disclosed in R.H.Foote (2002) in the summary of the challenge that is faced aspect use artificial insemination and the preservation sperm, American Society of Animal Science (http://www.asas.org/symposia/esupp2/Footehist.pdf) and " Reproduction in farm animals ", B.Hafez, E.S.E.Hafez. write the 7th edition, Philadelphia, Lippincott Williams ﹠amp; Wilkins, 2000.-X III is among the ISBN 0-683-30577-8 (ib.).In agricultural industry, produce this aspect two the animal breeding with particularly preferred genetic character and common animal, artificial insemination has all become a kind of mode of important, economic animal breeding.
Yet, have some limitation aspect the jenny gestation making by artificial insemination.For example, in the storage process, after thaw back (for the sperm of cryopreservation) and the insemination, the storage period of the sperm of being gathered and vigor are very important for successful breeding result.Whether available suitable preservation technology is arranged, and this is according to concrete animal species and different.For ox, the cryopreservation technology is widely used.On the contrary, for example the sperm of pig is lower to cryopreservation technology tolerance from other species, cause seminal fluid handle and store possibility aspect these flexibility lower.
Moreover in order to obtain to have the sperm of suitable fertility, employed method for preserving need make described sperm still keep fertility after insemination.Be subjected to pay close attention to very much and by big quantity research, promptly described storage method and mode can guarantee that sperm still keeps fertility in the long period and when insemination after collection with the method for preserving that provides storage method as described below and mode to be purpose.
If after insemination, keep the storage period aspect the fertility shorter, will be difficult to satisfy the preferred time point of insemination so with respect to ovulation.Under the situation of shorter storage period feature, the good sperm preservation technology of the fertility of longer time is vital to provide longer storage period to reach therefore.Now still do not have method for preserving can provide sufficiently long storage period and the sufficient sperm viability in the insemination sufficiently long time of back to satisfy the demand of breeder to flexibility---particularly when between male position (i.e. the place of therefore carrying out semen collection) and the female receptor position distance far away and consuming time being arranged.
Moreover, provide more controlled and will reduce the demand of carrying out artificial excitation's ovulation by HORMONE TREATMENT with method for preserving more long-acting sperm availability.This all will benefit aspect economic aspect (according to consumer's needs) and the animal health.
Therefore, need to guarantee that sperm fertility after insemination keeps the method for long period, keep the fertility of long period in the time of for example in placing female recipient's body.
Now, in ox, use the artificial insemination (AI) of the sperm of cryopreservation extensively to be carried out.The sperm of cryopreservation can be stored in many decades in the liquid nitrogen before use.Yet, after sperm thaws, must in several hours, carry out AI.The sperm of described cryopreservation has the fertilization potentiality and reaches about 12-24 hour after insemination, so must approximately carry out AI in 12-24 hour before ovulation.Therefore need the preservation technology that is used for the ox breeding as follows, promptly this technology can provide and have sufficiently long storage period feature and preferably keep fertility to reach the sperm of a couple of days.
In some country, the bull sperm that uses fluid storage is to reduce the number of spermatid in each AI dosage.Described sperm has fertility and reaches about 24-36 hour before insemination.AI must carry out in about 24 hours after the sperm collection.Yet the bull sperm of liquid preservation is limited because of multiple shortcoming, and for example storage period shortens/reduces and distribution reduces.
In pig, the sperm of cryopreservation only is used to specific purpose and for example exports, transports for long-distance and be used to control contagious disease.AI in the pig carries out with the seminal fluid of liquid preservation usually.The storage time of the seminal fluid of liquid preservation (sperm) will depend on uses for which kind of thinner.Sperm with fugitive thinner dilution keeps fertility to reach about 2-3 days, and keeps fertility to be up to 5-6 days with the sperm of long-acting thinner dilution.After insemination, the fertility of described sperm continues about 12-24 hour.Most sow inseminated twice with about 24 hours interval in oestrus.The sperm that provides storage time (for example 1 week) longer before the insemination and/or insemination back to prolong with system more flexibly stores and discharges (for example above 24 hours), the breeding industry will have more effectively output and distribution, and the breeder will reduce the requirement with respect to the accurate insemination selection of time of ovulating.
In the horse breeding, owing to lack available horse sperm preservation technology, the breeder depends on fresh sperm usually.This is a very serious problem in horse breeding and horse racing industry, and this is because preferred kind horse is usually located at different countries for specific broodmare, thereby needs long haulage time.In addition, owing to lack suitable horse sperm method for preserving, the AI of fresh horse sperm must finish in 24 hours after gathering described sperm.Therefore the horse breeding can be with disadvantageous time pressure, described pressure cause sperm quality to descend usually or since insemination become pregnant with inconsistent minimizing that ovulate.
Therefore, need method for preserving as follows, promptly described method for preserving can be guaranteed the longer storage period after relevant longer storage period of haulage time and insemination are passed in essential sending.Available horse seminal fluid method for preserving will have very big economic worth and practical value, and may cause the change of horse breeding industry.
In storage process, before the insemination and the sperm preservation system of enough vigor all can be provided all will be useful to general breeding industry after the insemination.Preservation system more flexibly will make the breeding work of all animal species be more prone to, and can increase successful becoming pregnant.Make the breeder not too depend on coupling and provide greater flexibility with respect to the system of most preferred insemination time point of ovulation.
For the fertility of the sperm that strengthens ejaculation, after deliberation multiple method for preserving comprise cryopreservation and liquid preservation.
Delivered the multinomial research that in capsule, stores sperm.The encapsulated method that forms a kind of particle has been used in these researchs, and promptly sperm is positioned at by semi-transparent membrane-enclosed liquid core in the heart in described particle.
Nebel et al. (1985), Microencapsulation of bovine spermatozoa.J.Anim.Sci.198560 (6): 1631-39 have described the method for a kind of capsule of making in conjunction with poly-D-lysine with alginates with the ox spermatophore.This method is based on from Lim and Sun (1980), Microencapsulated islets as bioartificial endocrine pancreas.Science 1980210 (4472): the former method of delivering of 908-10, this paper studies the encapsulated insulin of producing packing by langerhans cells.Nebel et al. (1985) has reported the result in 37 ℃ of following storages and 335 inseminations, and has compared the result of sperm that incapsulates and the control sample that does not incapsulate.This research has been reported between the sample that incapsulates and the control sample does not have big difference.Other researchs of delivering are also verified, can sperm be loaded in the capsule with similar method, and keep their functional (Munkittrick et al. (1992) Accessory sperm numbers for cattle inseminated with protaminesulfate microcapsules.J.Dairy Sci. in vivo, 75 (3): 725-31 (ox), Vishwanathet al. (1997) Selected times of insemination with microencapsulatedbovine spermatozoa affect pregnancy rates of synchronized heifers.Theriogenology, 48:369-76 (ox) and 5 ℃ of .Reprod.Dom.Anim. of Maxwell et al. (1996) .Survivaland fertility of micro-encapsulated ram spermatozoa stored at, 31:665-73 (sheep)).Yet Munkittrick et al. (1992) and Vishwanath et al. (1997) reported, when the sperm that incapsulates and untreated sperm were inseminated simultaneously, it was renderd a service not as the latter.This can be interpreted as, and the sperm that incapsulates may need some times to discharge from capsule in prefecundation.
Conte et al. (1998) is in EP 0922 451B1 that licenses to Universit à di Pavia and Universit à Degli Studi Di Milano and Torre et al. (2002), Boar semen controlled delivery system:storage and in vitrospermatozoa release.J.Contol.Release, 85:83-89 have developed the another kind of method that the boar sperm is incapsulated.In the method, sperm is added in the solution of calcic or barium, and this suspension is splashed in the solution of alginate-containing.When drop runs into alginate soln, around them, be formed naturally the capsule of calcium alginate or Barium alginate.Compare with the method that Nebel et al. (1985) describes, this method is claimed that pair cell is gentle more.Another advantage is, this method can be diluted sperm solution hardly, and this is claimed the vigor that helps described cell.
Faustini et al. (2004), 18 ℃ of Boar spermatozoa encapsulated in bariumalginate membranes:a microdensitometric evaluation of someenzymatic activities during storage at, Theriogenology, 61 (1): 173-184 has reported that the sperm of remarkable vast scale has complete acrosome, and compare with the sperm that is untreated that stores under the same conditions, the sperm that incapsulates storage with this method has enzyme leakage still less.
At nearest paper Weber et al (2006), Design ofhigh-throughput-compatible protocols for microencapsulation, cryopreservation and release of bovine spermatozoa.Journ.Biotechnol. has also described a kind of new system that Niu Jingzi is incapsulated of being used among the 123:155-163.This system is designed to the sperm that high flux ground preparation incapsulates, use be poly-diallyl alkyl dimethyl ammonium chloride (pDADMAC) capsule of Ca-alginates or sulfate cellulose.
At last, people's such as Chou US 6,596,310B1 (2003) disclose a kind of by periodically discharge the artificial insemination method of sperm from capsule or solid beads, wherein owing to used the energy of not supporting capacitation (capacitation), described sperm is maintained at a kind of state of non-capacitation.
Summary of the invention
The present invention is based on such accident and find, be about to sperm and be embedded in that (wherein said biopolymer substrate is made up of the alginates that are rich in guluronic acid) causes sperm having more excellent preservation characteristic aspect storage period, vigor and the fertilization in the biopolymer substrate.Described biopolymerization composition granule and in sperm therefore can be used in the artificial insemination of animal.
A non-limiting advantage of sperm preservation biopolymerization objects system of the present invention is it by the fertility of longer time of giving described sperm after insemination, and therefore makes the described insemination time so not most important and obtain benefit with respect to ovulation.
Biopolymerization composition granule according to the present invention can be used for animal breeding and the production in the agricultural industry.Therefore, be that described biopolymerization composition granule is useful when obtaining to have the animal of feature of special needs in target.In target is that described biopolymerization composition granule also is useful when producing conventional animal (for example producing beef cattle).
According to the present invention, if described biopolymerization composition granule (for example alginates particle) can be in the receptor animal dissolving under the physiological condition of (promptly in uterus or uterine neck), so described biopolymerization composition granule can directly use in described receptor animal and inseminate after this manner.Therefore, biopolymerization composition granule according to the present invention can make described sperm controllably discharge.Before with described sperm insemination, also can be with the stripping from described biopolymerization composition granule of described sperm.
As indicated above, prior art mainly shows and sperm can be wrapped in the biopolymerization composite capsule, and the liquid core that wherein said sperm is comprised in the biopolymerization composite capsule in the heart.Only (people's such as Chou US 6,596 310B1) has mentioned sperm has been embedded in possibility in the solid polymer substrate, but do not provided the example of the solid beads described in the literary composition for one piece of list of references.
The inventor has found a kind of method of different fixedly sperm in addition.According to the present invention, described sperm is embedded in the solid gel network that is prepared by alginate jelly, and wherein employed alginates are rich in guluronic acid.The inventor has been found that disclosed comparing with capsule and pearl packing has a plurality of advantages in immobilization and the prior art in the solid gel network.
Do not rigidly adhere in concrete theory, the inventor thinks that by using biopolymerization composition granule and method of the present invention, the embedding meeting causes immobilization, and for example the natural motion of described sperm is owing to the constraint of described gel network is restricted.Owing to the high concentration cell is limited with the physical motion that exists high-viscosity polymer to cause may be to influence sperm to survive in cauda epididymidis and one of its functional factor of keeping (Watson 1993).Sperm can be stored (Watson 1993) by long-time (surpassing for 1 week) in cauda epididymidis.The application of being rich in the alginates of guluronic acid makes the biopolymerization composition granule of the sperm might obtain to comprise embedding, and described biopolymerization composition granule can be used as the preservation system in practice, and can be used for artificial insemination---for example animal breeding.
Therefore, according to an aspect, the invention provides the biopolymerization composition granule that is used for the preservation sperm, wherein said sperm is embedded in the biopolymer gel network, and wherein the described biopolymerization composition granule of the described sperm of embedding comprises the alginates that are rich in guluronic acid.
According to another aspect of the present invention, the described biopolymer of the described sperm of embedding comprises calcium alginate.
According on the other hand, described biopolymer comprises low viscous alginates.
Also according to a further aspect in the invention, the concentration of the described alginates in biopolymerization composition granule of the present invention is at least 0.1%.
Also according to a further aspect in the invention, the concentration of the described alginates in biopolymerization composition granule of the present invention is at least 0.1% to 6% alginates.
Also according to a further aspect in the invention, the concentration of the described alginates in biopolymerization composition granule of the present invention is at least 1%.
In addition, according to an aspect of the present invention, the concentration of the described sperm in described biopolymerization composition granule is at least 0.1 * 10
6Sperm/ml, for example at least 100 * 10
6Sperm/ml, as the upper limit at least 2.5 * 10
9Sperm/ml.
Also according on the other hand, particle of the present invention can be stored in the solution, and for example wherein said storage liquid: the biopolymer proportion of particles is at least 1: 1 to 1: 100.
Also basis on the other hand; described sperm can with compound or the reagent altogether embedding useful to fertility and/or animal health, for example one or more described compound or reagent are selected from thinner, cryoprotector, antibiotic, antibody, antioxidant, albumen and hormone without limitation.
According to another aspect of the present invention, described sperm be selected from altogether embedding of one or more following antioxidant: acetonate, 2,2,6,6-tetramethyl-piperidyl-1-oxygen, 4-hydroxyl-2,2,6,6-tetramethyl-piperidyl-1-oxygen (4-hydroksy-2,2,6,6-tetra-metyl-peperidin-1-oxyl), superoxide dismutase, catalase, glutathione peroxidase (glutathionperoxydase), Butylated Hydroxytoluene and Butylated Hydroxyanisole (butylated hydroxyanisol).
According to an aspect, described biopolymerization composition granule is coated.Described dressing can be selected from poly-D-lysine (polylysin), shitosan, sulfate cellulose, hydroxypropyl methylcellulose and diallyl dimethyl ammoniumchloride without limitation.
Biopolymerization composition granule of the present invention comprises the sperm of gathering from be selected from following animal without limitation: for example purebred dog of pig, ox, horse, sheep, goat, rabbit, poultry, pet, aquatic animal and animals on the brink of extinction kind, preferred pig, ox, fleece animal and horse.Therefore, biopolymerization composition granule of the present invention can be used for artificial insemination method by routine for example AI, IVF and ICSI obtains gestation in the jenny of subordinate.
According to an aspect of the present invention, the sperm that is used to form described particle is comprised in the seminal fluid.
Randomly, described biopolymerization composition granule can further be handled through dehydration, cryopreservation or freeze drying.
The present invention also provides a kind of method for preparing particle of the present invention, wherein is rich in the alginates of guluronic acid and the mixture of sperm and dropwise is added in the gelling soln.According to an aspect of the present invention, described gelling soln comprises and is selected from one or more following ions without limitation: calcium, sodium, barium and magnesium, preferred calcium and sodium ion.
In addition, described biopolymerization composition granule can randomly directly form in the seminal fluid container.
An aspect of the method according to this invention, described particle can further be handled through dehydration, cryopreservation or freeze drying.
The present invention also provides the purposes of biopolymerization composition granule of the present invention in animal breeding, and described animal is for example purebred dog of pig, ox, horse, sheep, goat, rabbit, poultry, pet, fleece animal, aquatic animal and animals on the brink of extinction kind for example.According to an aspect of the present invention, described biopolymerization composition granule is used for the breeding of pig, ox or horse.According to an aspect, described particle is directly inseminated.Also according to another aspect, described particle is dissolved before insemination.Also according on the other hand, particle of the present invention uses simultaneously with free sperm, fixing sperm.
At last, the invention provides a kind of method that is used to make the animal fertilization, the sperm that wherein is preserved in the particle of the present invention is imported in the receptor jenny.
Description of drawings
Fig. 1 demonstrates ox (figure A) and boar (figure B) the sperm external storage of (20 ℃ of oxen, 18 ℃ of boars) at ambient temperature.At the reference sample with have in the sample of fixing sperm, the motility rate value provides with the functional form in storage time.
Fig. 2 demonstrates Niu Jingzi (figure A) and the external storage of boar sperm (figure B) under 37 ℃.At the reference sample with have in the sample of fixing sperm, the motility rate value provides with the functional form in storage time.
Fig. 3 demonstrates the external storage of (18 ℃) at ambient temperature of boar sperm.At the reference sample with have in the sample of fixing sperm, the motility rate value provides with the functional form in storage time.In described storage experiment, used the storage medium (with respect to the amount of pearl) of different amounts, identify as serial title.Described sperm is with 1 * 10
9The concentration of sperm/ml is fixed on the pearl that diameter is 1mm.
Fig. 4: the boar sperm is the external storage of (18 ℃) at ambient temperature.Motility rate value with fixing sample of sperm provides with the functional form in storage time.Pearl with fixedly sperm of variable concentrations is used to store in the experiment, identifies as serial title.Described sperm is fixed on the pearl that diameter is 3mm.
Embodiment
As mentioned before, the focus of studying in the past is primarily aimed at sperm is incapsulated to help in vitro fertilization and artificial insemination.The method that the inventor has adopted a kind of fundamental difference is with fixing sperm, wherein said sperm is embedded in the biopolymer network of gel or solid gel granule interior, and wherein said biopolymer network is made up of the alginates that are rich in guluronic acid.That describes in the prior art before this method is different from basically incapsulates sperm, and sperm is comprised in the capsule with liquid core in the latter, can move about as ground in its natural surroundings at the described in the heart sperm of described liquid core.The described biopolymerization composition granule of embedding sperm of the present invention does not rely on use can guarantee that described sperm maintains a kind of solution of non-capacitation state.
Term used herein " embedding " or " embedding " are construed as fixedly sperm, thereby make described sperm lose the possibility of its natural motion that has.Described fixing degree will depend on the characteristic (for example mechanical strength) of biopolymerization composition granule and the type of the polymer that uses and changing.Yet, it will be appreciated that, be embedded in described sperm in the biopolymerization composition granule of the present invention and lose the possibility of the natural motion that they have, can exist and this possibility is stored in the liquid (for example in the liquid core of capsule in the heart) time at described sperm.
Term used herein " biopolymerization composition granule " is meant a kind of like this particle, promptly can reduce the possibility of sperm motility when it comprises.Biopolymerization composition granule of the present invention is made of a kind of like this material, and promptly this material forms the network of being made up of the alginate jelly that is rich in guluronic acid.Described biopolymerization composition granule does not rely on the 3D shape of biopolymerization composition granule of the present invention in the function aspect the preservation sperm.Therefore, described biopolymerization composition granule of the present invention can have different shapes, and is for example spherical or cylindrical.
Alginates are a kind of polymer of being made up of guluronic acid (G) and mannuronic acid (M).Alginates are total compositions of the cell wall of all brown seaweed kinds (Phaeophyceae (Phaeophyceae)), and common available alkaline solution extracts.The ratio of mannuronic acid and guluronic acid (M/G) is according to concrete alginates plant (alginophyte) (producing the marine algas of alginates) and different seasons and marked change in alginates.
Alginates also can be by pseudomonad and synthetic (the Svanem et al. of azotobacter, (2001), TheJournal of Biological Chemistry, Vol 276:34,31542-31550, Jain etal., (2003), Molecular Microbiology, 47 (4), 1123-1133, Scott andQuatrano (1982), Applied and Environmental Microbiology, 44:3,754-756).
Alginates for example be widely used in food industries (as stabilizing agent and be used to control viscosity), in pharmacy and the cosmetics industry (as disintegrant).Can obtain to be rich in alginates (the Mancini et al. of guluronic acid or mannuronic acid respectively based on different purposes, (1999), Journal of Food Engineering 39,369-378), and the multiple method that is used to produce the alginates that are rich in guluronic acid is known (referring to WO 8603781, US 4,990,601, US 5,639,467).
Term used herein " is rich in the alginates of guluronic acid " or " being rich in the alginates of G " is meant that alginates contain the amount height of the amount of guluronic acid than mannuronic acid in the polymer chain that constitutes described polysaccharide.Be used for understanding the example that " is rich in G " as the normally used term of those skilled in the art, can be apparent from top two sections described prior aries.
Because bivalent cation is Ca for example
2+And the interaction between the section structure of guluronic acid has formed alginate jelly in the alginate polymer chain.Therefore, the formation of described alginate jelly can be finished under condition as mild as a dove, and is suitable for fixed cell.Therefore, alginate jelly is normally used for fixing polytype cell.Yet, the fixing the most frequently used starting point that forms polytype capsule subsequently in alginates with liquid core.
When mentioning alginates, alginate jelly, alginates network or biopolymerization composition granule of the present invention in this article, should understand described alginates, alginate jelly, alginates particle or biopolymer network of the present invention or particle and be rich in guluronic acid.
The limiting examples of suitable alginates type is from FMC Biopolymer AS, Drammen, and FMC LF 10/40, FMC LF 10/60 and FMC LF 20/60 that Norway obtains, or from from Sigma, Oslo, the A2033 that Norway obtains.
Term used herein " storage period " be meant sperm before insemination external storage and in the time that in receptor's animal body, all maintains fertility after the insemination.The concrete storage period of the sperm of separate sources can be different.Yet after the sperm that conventional storage method is inseminated after storing is compared, this preservation system (wherein sperm is embedded in the biopolymerization composition granule of the present invention) has obtained the storage period after the longer insemination with semen collection.According to an example of the present invention, the pig sperm is more preferably reaching after the insemination more than 24 hours keeping good fertility to reach more than 12 hours after the insemination.According to another example of the present invention, bull sperm is more preferably reaching after the insemination more than 24 hours keeping good fertility to reach more than 12 hours after the insemination.
Term used herein " breeding " is meant any method that is used to obtain jenny gestation.A non-limiting tabulation of this method comprises: IVF, artificial insemination and ICSI.In addition, the animal of the proterties that obtains to have special needs and the breeding of commercially producing this two aspect are contained in breeding.
Term used herein " sperm " comprises sperm itself, and comprises the sperm that is included in the seminal fluid, promptly can directly use seminal fluid when forming biopolymer of the present invention.Yet the sperm that separates from seminal fluid (randomly being comprised in other suitable storage liquid) also can be used to form biopolymerization composition granule of the present invention.
Term used herein " seminal fluid container " comprises all types of package bodies that are suitable for preserving and storing sperm.
Former encapsulated method disclosed and as indicated above in the prior art is required great effort very much, and described immobilization operation generally comprises a plurality of steps.Yet biopolymerization composition granule according to the present invention can prepare with single stepping.More accurately, the calcium alginate particle that comprises sperm is easy to form by this way, promptly the described cell that is fixed is had MIN physiological stress and chemistry stress.The alginates particle can be prepared to multiple size and dimension, and the concentration of available multiple alginates type, concentration of alginate and the sperm that is fixed is prepared.
For embedding sperm of the present invention, using alginates is specially suitable as bio-polymer material, this is due to the fact that i) alginates can form gel under temperate condition, ii) alginates all are nontoxic for sperm and receptor animal, and iii) alginates can dissolve under physiological condition and therefore can discharge sperm in uterus or uterine neck.
According to an aspect of the present invention, can prepare described alginates particle by such mode, the solution that is about to comprise alginates and sperm dropwise adds in the gelling soln, the alginates pearl of described sperm that formed wherein embedding.
The preparation of alginates pearl is well known by persons skilled in the art, for example
O.and
G. (1990) Alginate as immobilization matrix forcells.Trends in Biotechnology 8, and is disclosed among 71-78 and the US 6,497,902.
Multiple gel solution can be applicable to form the biopolymerization composition granule, for example the alginates pearl.The solution of the various biopolymerization composition granules of multiple suitable formation is well known to those skilled in the art.According to the present invention, divalent ion (being also referred to as the gelling ion in this article) for example calcium and barium can be used for forming the alginates particle, for example to realize the gelation of described alginates.Alginates can form gel under the situation that has multiple divalent ion and multivalent ion.The use of calcium ion has caused quick-gelatinizing and has formed stronger particle relatively.On the other hand, the use of barium ions has formed even stronger particle, and this particle is more stable under physiological condition.It will be appreciated that, by the type that forms the employed gelling ion of biopolymerization composition granule of the present invention and amount can according to the required intensity of formed gel, the guluronic acid content of the type of use alginates and concentration, alginates and G-section length, the types and sources, the type (for example antibiotic, thinner, antioxidant etc.) etc. that is incorporated into other reagent in the described particle of use sperm change; And it will be appreciated that this change all within the scope of the present invention.Based on the instruction of prior art and this specification, those skilled in the art do not need too much work to identify and determine and can be embedded in all cpds in the biopolymerization composition granule of the present invention altogether with described sperm.
According to an aspect of the present invention, calcium ion and sodium ion are used to form the biopolymerization composition granule.Therefore, by changing the degree of concentration of alginate and crosslinked combination, can obtain having the particle of multiple sperm release characteristics, described particle is suitable for ovulation condition specific in for example specific animal kind or the multiple animal.
In addition, the mechanical property of the described biopolymerization composition granule of the concentration affects of described alginates, therefore and also influence the dissolution characteristic of described particle.Therefore, described concentration of alginate can change according to depending on the needed dissolution characteristic of for example described receptor animal under every kind of situation.Those skilled in the art do not need too much work can determine the various suitable concentration of the alginates that are rich in G that will use based on its general knowledge and based on the instruction of this paper when preparation biopolymerization composition granule of the present invention.According to an aspect of the present invention, the concentration of alginate in the biopolymerization composition granule of the present invention is at least 0.1 to 6%.
The sperm concentration that is embedded in the described biopolymerization composition granule of the present invention can change, this depend on sperm for example type/source, kind, receptor animal, insemination techniques or system, fertilization technology or system, be included in the existence of other reagent (antibody, antioxidant, thinner, albumen etc.) in the described particle.Described sperm concentration does not have lower limit and this concentration to change in principle, and this depends on dissolution characteristic of the source of for example described fertilization method, described sperm, described receptor animal, described biopolymerization composition granule etc.Those skilled in the art are according to the instruction and the general knowledge of this specification, based on the source of required fertilization method, sperm, receptor animal etc., do not need too much experiment need in each case can to determine the suitable sperm concentration that uses.Need to understand, when ICSI is used as required insemination method, can usage ratio such as the lower sperm of artificial insemination.
According to an aspect of the present invention, therefore described sperm concentration is at least 0.1 * 10
6Sperm/ml.
Discovery result of the present invention shows that for some animal, the survival rate of described sperm improves a lot with the increase of sperm concentration.Therefore, according to an example of the present invention, at least 2.5 * 10
9Pig sperm/ml is embedded in (referring to Fig. 4) in the biopolymerization composition granule.Yet, 100 * 10
6Pig sperm/ml also can make in described jenny gestation.For bull sperm, the applicable concentration range identical with the pig sperm.
By changing, can further change the concentration of described sperm in described biopolymerization composition granule with respect to the sperm of the alginates amount before the gelation or by before mixing described alginate soln and comprising the solution of sperm, concentrating or diluting described sperm solution.Yet, because the change of described sperm finally can cause the change of the concentration of alginates in resultant particle, keep the concentration of described alginates in described biopolymerization composition granule if desired, then must adjust the concentration of alginates in described initial alginate soln.Because in fact the high viscosity of alginates in solution may be difficult to use the alginate soln that contains above the 4-6% alginates.Therefore, according to an aspect of the present invention, described biopolymerization composition granule is made of low viscous alginates.
In addition, the sperm that is embedded in the described biopolymerization composition granule of the present invention is separated with surrounding environment to a certain extent, and just is released until the calcium alginate gel network dissolves.In addition, described calcium alginate gel network is slowly dissolving under physiological condition, and this can cause that sperm discharges lentamente on described immobilization matrix.Because this effect, can there be the longer time in the sperm with fertility.On described immobilization matrix, use dissimilar dressing controlled dissolution rates.Multiple useful dressing is known, includes but not limited to poly-D-lysine, shitosan, sulfate fiber, hydroxypropyl methylcellulose or diallyl dimethyl ammoniumchloride.
In order further to improve the sperm quality, can be with described sperm and various to sperm survival and the very important solution embedding altogether of vigor.According to one embodiment of the invention, comprise thinner in the described biopolymerization composition granule of the present invention.
The sperm thinner is normally used for a plurality of purposes:
-increase volume
Sperm quantity in the-standardized A I dosage
-cushioning effect
The osmolarity of-acquisition physiological environment
-provide nutritional support for described spermatid
The pollution of-controlling microbial (antibiotic), perhaps under the situation of cryopreservation:
-protection spermatid is avoided freezing-thawing damage.
Require and international legislation based on the type (source) of for example sperm, species, method for preserving, storage temperature, insemination method, veterinary hygiene, those skilled in the art know the suitable diluent of how selecting to can be used in the biopolymerization composition granule of the present invention.Breast thinner and TRIS only are the examples of the several non-limiting in a large amount of commercially available sperm thinners that are suitable for.Another kind of useful thinner is Aalbers, JG, Rademaker, JHM, Grooten, HJG and Johnson, LA, 1983:Fecundity of boar semen stored in BTS, Kiev, Zorlesco and Modenaextenders under field conditions.J.Anim.Sci.75 (Suppl.1), 314-315 and Aalbers, JG, Johnson, LA, Rademaker, JHM and Grooten HJG, 1984:Use of boar spermatozoa for AI:fertility and morphology ofsemen diluted in BTS and used for insemination within 24 hrs or 25to 48 hrs after collection.Proc.10th Int Congr.Anim.Reprod andArtif.Insemin.Urbana IL, Vol II, No 180, the thinner of reporting among the pp 1-3 is commonly referred to as BTS.BTS can buy from Minit ü b i Tyskland.(Minitüb?Abfüll-und?Labortechnik?GmbH?&?Co.KG，Hauptstrasse?41，DE-84184Tiefenbach，Germany)。
Also another kind of useful thinner is can be from IMV (Instrument de MedecineVeterinaire, 10, rue Georges Clemenceau, B.P.81, FR-61302 L ' AIGLECedex, the Tri X-Cell thinner of France) buying.
The composition of described various thinners, pH, buffer capacity, osmolarity and antibiotic are different.Many is disclosed, and other are business secrets.Only for example lactose and glucose constitute the simplest thinner by different sugar juices.Be appreciated that according to the present invention based on the technical staff's in sperm preservation field general knowledge and can use plurality of diluent.The type of thinner used according to the invention can be different, and this depends on the type, method for preserving, storage temperature etc. of the source of described sperm, the polymer that uses.Under the situation that does not break away from the disclosed invention scope of this specification and the appended claims, how to determine and select the type of employed thinner or appropriate amount in those skilled in the art's ken.
Biopolymerization composition granule according to the present invention can form with multiple size according to method well known in the prior art.Which kind of size suits, and this depends on multiple factor, for example the size and dimension of the type of sperm source, employed fertilization device and size, the sperm container that uses etc.Biopolymerization composition granule according to the present invention can form with the diameter that is suitable for insemination techniques all in the prior art, so that easy operating, processing, transportation and preservation.
According to a further aspect of the invention, described biopolymerization composition granule forms in container (tubule of for example inseminating), forms the particle that adapts fully with described container dimensional.Therefore, the situation for form described biopolymerization composition granule in container can not need other storage liquid.
According to an aspect of the present invention, described biopolymerization composition granule comprises the sperm of gathering from ox.According to another aspect of the present invention, biopolymerization composition granule of the present invention comprises the sperm of gathering from pig.
Though to comprise the alginates particle of gathering respectively from the sperm of ox and pig the present invention has been described, described biopolymerization composition granule can also embedding from the sperm of other animal kinds.Without limitation, all right embedding collection of described biopolymerization composition granule is from for example sperm of for example purebred dog of horse, sheep, goat, rabbit, poultry, pet, aquatic animal and multiple endangered species.
In addition, in any case do not represent, thus biopolymerization composition granule of the present invention can not be used for preservation people sperm and be used for sterile treatment.Therefore, need to understand described term used herein " animal " and also contain the people.
In addition, in the aquaculture industry, also extensively need the sperm preservation technology.Therefore, the sperm that is derived from aquatic animal also can be embedded in the described biopolymerization composition granule of the present invention, so this class animal also is included in the term used herein " animal ".
Sperm is fixed in the biopolymer gel network also can be used for---even in female reproductive organs inside---and produces storing the favourable microenvironment of sperm.This can realize favourable common encapsulating solution such as described sperm, fertilization degree, animal health with other.The limiting examples of the material of for example, invigorating in storage process has antioxidant or albumen, hormone etc.Described sperm can also with other reagent or the compound altogether embedding useful to fertility or animal health.This reagent or compound can be for example can be used for controlling or antibiotic (as streptomycin, neomycin, gentamicin, penicillin, lincomycin, spectinomycin, Amoxicillin, tylosin etc.), cryoprotector, antibody, the hormone of prevention of STD or the compound that increases reproductive efficiency (as from US 5; 972,592 compounds of knowing).
Therefore, also according to another aspect of the present invention, described biopolymerization composition granule also comprises fertility or the useful compound of animal health, for example composition of antioxidant, antibiotic, antibody, hormone or reproductive efficiency promoter or above-mentioned substance except comprising sperm.According to an embodiment preferred of the present invention, described biopolymerization composition granule comprises for example acetonate of antioxidant.
Described biopolymerization composition granule of the present invention can randomly be stored in the known sperm storage liquid, for example newborn thinner of described solution, PBS (phosphate buffered saline), BTS, Tri X-Cell, EDTA-thinner, Kiev-thinner, Modena, MR-A, Androhep, Acromax, Triladyl
, Biladyl
, Bioxcell, Biociphos plus etc., contain or do not contain for example acetonate, 2 of antioxidant, 2,6,6-tetramethyl-piperidyl-1-oxygen (abbreviating Tempo usually as), 4- hydroxyl 2,2,6,6-tetramethyl-piperidyl-1-oxygen (abbreviating Tempol usually as), superoxide dismutase, catalase, glutathione peroxidase, Butylated Hydroxytoluene (abbreviating BHT usually as) and Butylated Hydroxyanisole (abbreviating BHA usually as).The selection of storage liquid is not crucial, as long as described storage liquid does not comprise the compound of the described biopolymer particle characteristics of possibility negative effect.For example, for the alginates particle, described storage liquid should not comprise too many in conjunction with divalent ion and therefore dissolve the chelating agent of described gel.
In storage process, the biopolymerization composition granule can change with respect to the ratio of described storage liquid, and this depends on the type of the alginates that are rich in G and concentration, the source of sperm, the insemination method etc. of using.Embodiment according to the present invention, described storage liquid: the biopolymer proportion of particles is at least 1: 1, for example at least 1: 2, at least 1: 3, at least 1: 4, at least 1: 5, at least 1: 6, at least 1: 7, at least 1: 8, at least 1: 9 or at least 1: 10.Can SC service ceiling according to the present invention at least 1: 100 storage liquid: biopolymerization composition granule ratio.
In addition, described biopolymerization composition granule of the present invention can randomly be stored under room temperature (for example 18 ℃ or 20 ℃), physiological temp (37 ℃) or the cold conditions (for example 5 ℃).In addition, described biopolymerization composition granule can further be handled through dehydration, cryopreservation or freeze drying.For the situation of cryopreservation, before insemination, then described biopolymerization composition granule is thawed.
Usually so that the sperm of liquid preservation is in being embedded in biopolymerization composition granule of the present invention, can be stored longer a period of time.According to another embodiment of the invention, described biopolymerization composition granule is directly used in inseminates to the receptor animal.Randomly, can be before insemination with described biopolymerization composition granule dissolving to discharge described sperm.Yet according to another embodiment, described biopolymerization composition granule can also or be used in combination with free sperm.
Though be described in conjunction with a specific embodiment thereof the present invention, should have understood and to do further change to the present invention.The application is intended to contain any variant of the present invention, application or selection, these are usually according to principle of the present invention and comprise the variation scheme of this paper disclosure, described variation scheme belongs to known habitual practice in the technical field of the invention, and applicable to the essential characteristic in the disclosed and claim scope of enclosing above.
Preamble has disclosed general aspects of the present invention to the description of many aspects of the present invention, and those skilled in the art can be by the general knowledge (content that comprises the list of references of quoting herein) in utilization artificial insemination and the breeding technical field based on various application, and the scope that does not need a large amount of experiments and do not depart from purport of the present invention and appended claims can easily improve and/or change biopolymerization composition granule of the present invention and their preparation and purposes.Therefore, based on instruction and guidance that this paper provides, this change or improvement are intended in the intended scope of the equivalence of embodiment disclosed herein.Need to understand, term used herein is in order to describe rather than purpose in order to limit.Therefore, the term of this specification can be made an explanation by instruction and the guidance that those skilled in the art provide according to this paper in conjunction with the basis of its knowledge.
Following non-limiting example will further be explained the present invention.
Embodiment
Material and method
Material
Used following chemical reagent: from Riedel de
(Seelze, CaCl Germany)
2H
2O, K
2HPO
4, NaH
2PO
4, NaCl and sodium citrate; From NorskMedisinaldepot (Oslo, Dextrose Monohydrate Norway); FMC Biopolymer A/S (Drammen, the mosanom that Norway) provides (PROTANAL LF 10/60);
The sperm source
The ox sperm exists
Trondheim, the Geno experiment (Genofacility) of Norway is gathered.The boar sperm is at Hamar, and gather the Norsvin of Norway experiment field.After ejaculation, soon Niu Jingzi is diluted in the newborn thinner with 1+2.The boar sperm with 1+4 be diluted in TRI X-CELL (IMV Technologies, L ' Aigle Cedex, France) in.The selection of this dilution factor or dilution buffer liquid is not vital for described immobilization operation or the long-term surviving of described sperm after being fixed, and only is in order to help described sperm to be stored until being further processed.
Buffer solution
Used following medium.The IVT:3g 1 of improvement
-1Glucose, 20g 1
-1Sodium citrate, 2.1g 1
-1NaHCO
3, 1.16g 1
-1NaCl, 3g 1
-1EDTA, pH 7.35.BTS (containing 3mM Ca): 37g 1
-1Glucose, 0.44g 1
-1CaCl
2, 4.5g 1
-1Sodium citrate, 1.25g 1
-1NaHCO
3, 1.25g 1
-1EDTA, 0.74g 1
-1KCl, 1g 1
-1Neomycin, pH 7.2.Pearl gelling soln: 7.3g 1
-1CaCl
2, 5.96g 1
-1NaCl.Breast thinner: 110g 1
-1Molico skimmed milk, 120ml 1
-1Yolk, 6.25g 1
-1Streptomycin, 1.5 hundred ten thousand (mill) I.E 1
-1Penicillin.
Fixed cell
Geno or a Norsvin experiment collection spermoblast and as above-mentioned the dilution.Before fixing with described sperm through centrifugal concentrated (800g, 20min, 20 ℃).The amount of the supernatant of removing in every batch is decided according to required sperm concentration in the finished product pearl.After removing unnecessary supernatant, cell precipitation is suspended lightly again by gentle jolting.Then the aseptic solution of sodium alginate of described cell suspension and 6% (w/v) is mixed lightly.With described alginate soln with 2 parts of cell suspensions than the volume ratio of 1 part of alginate soln add (after deliberation the various combination of volume ratio of a large amount of concentration of alginate, alginate soln and cell suspension.What this paper provided is typical numerical value and operation).
The mixture of alginates and cell dropwise is added in the pearl gelling soln (sees above).In order to produce the pearl with 3mm diameter, the syringe needle by internal diameter 0.5mm adds described solution.In order to produce the more pearl of minor diameter (little) to diameter less than 1mm, used a kind of system, described system is used the size that forms droplet on the needle point based on using air-flow to be limited in.In described gelling soln, use sodium chloride be for guarantee the concentration of polysaccharide in all pearls evenly (
O.and
G. (1990) Alginate as immobilization matrix forcells.Trends in Biotechnology 8,71-78).In order to limit the Ca in the described pearl
2+Measure, described pearl is stirred in described pearl gelling soln be no more than 8min.Carry out whole immobilization operation at ambient temperature.
The sperm that external at ambient temperature storage is fixing
A purpose of immobilization operation is to prolong the time that described sperm can store before insemination.Therefore carried out the experiment of the described sperm of external storage in buffer solution at ambient temperature.In all experiments, all comprise reference sample from the on-fixed sperm of homogeneous ejaculation.Measure the quality that motility rate is assessed described sperm by different time points at storage process.At each time point, analytic sample also writes down the motility rate of described fixedly sperm and the motility rate of control sample.For with described experimental system optimization, after deliberation a large amount of combinations (data not shown) of reservoir volume and cell density.Experiment condition described below is general, and is used in the experiment of hereinafter reported.
With in the experiment of Niu Jingzi, the pearl that about 2ml is fixed with sperm is transferred in the 13ml centrifuge tube and adds newborn diluent solution with the volume ratio of 1+2.Prepared control sample (in all pipes, using the volume of about 6ml) by total dilution factor in the centrifuge tube of 13ml with free sperm that suspends with 1+15.In the former experiment, having demonstrated this dilution factor almost is best (data not shown) for the maintenance of motility rate in the described reference sample storage process.
In experiment, under 18 ℃, described sperm is stored among the BTS (containing 3mM Ca) with the boar sperm.Hereinafter in Bao Dao the experiment, in all experiments, dilute described control sample with 1+10, obtain 20ml in the 50ml centrifuge tube cumulative volume (before demonstrated this dilution factor for storage process in the maintenance of motility rate be best).To comprise that the fixing pearl of sperm is transferred in the 50ml centrifuge tube and add BTS and (contain 3mM CaCl with the ratio of 1+3
2).Before being transferred to storage liquid, comprise the fixedly pearl of sperm with described storage liquid flushing.
Store fixing sperm down at 37 ℃
In order to assess the survival rate of described sperm under physiological temp, under 37 ℃, be stored in the motility rate that has write down described sperm in the process of sperm.Before the storage experiment under 37 ℃ Niu Jingzi is transferred in the buffer solution.As indicated above, comprised reference sample in each experiment from the homogeneous ejaculation.In the centrifuge tube of 13ml, carried out described test with every pipe 8ml cumulative volume.The reference sample is diluted in the storage liquid with 1+4.For fixing sperm, the pearl that 3ml is comprised sperm adds in the 6ml buffer solution and is stored in the 13ml centrifuge tube.Use similar mode to handle the boar sperm, difference has been to use conventional BTS (containing 3mM Ca).
The assessment motility rate
Estimate the motility rate of the described sperm of assessment by microscope.Sampling from described reservoir vessel (general 1ml) also is transferred in the 1.5ml Eppendorf pipe.Before assessment with minimum 15 minutes of the preheating in 37 ℃ micro-constant temperature instrument (heat-block) of described pipe.When measuring, 2.5 μ l samples are added on the microslide of preheating, use light microscopy then immediately.Estimate to have in each sample the sperm quantity of mobility with 5% interval up and down.If actual capabilities, which kind of sample does not allow operating personnel know in evaluation process is.
Before estimating motility rate, fixing sperm must be discharged from described pearl.In order to dissolve described alginates pearl, in the centrifuge tube of 13ml, 1 part of pearl is added in 3 parts of IVT solution.Before assessment, described pipe is placed on the test tube circulator (tube-tumbler) then, make it to mix about 30min.
Result and discussion
The fixing survival rate of sperm
For Niu Jingzi and boar sperm, the data that store experiment from reference sample (containing revocable sperm) and the sperm that contains the fixing sample of sperm have been shown among Fig. 1.For the boar sperm, data have been shown from the pearl of 2 kinds of different sizes.Its reason is that in order to use the pearl that is suitable for carrying out with existing apparatus intrauterine insemination, the size of described pearl must be less than 1mm.
Can be observed as the data that from Fig. 1, present, as if described fixing sperm have after immobilization soon than the low slightly motility rate of sperm in the described reference sample.The difference of this motility rate can be caused by described immobilization operation or described dissolving operation, because must will comprise the fixedly described pearl dissolving of sperm before the motility rate assessment.Yet, before insemination, do not need described pearl dissolving, because described pearl can dissolving (data not shown) at leisure under physiological condition.
Motility rate difference between reference sample and the fixing sperm can be dwindled in storage process, as if this is because the motility rate in the described reference sample is than descending soon in the described fixed sample, especially for the boar sperm.For fixing boar sperm, in this concrete experiment, storage records about 50% motility rate after 5 days in the pearl of 3mm diameter.At this moment, in the reference sample, record 10% motility rate.These results show that immobilization influences the feasible ability that helps their tolerance external storages under 18 ℃ of mode of the local environment of cell or described cell.
For Niu Jingzi, the motility rate between described reference sample and the described fixing sperm shown in later stage of experiment do not have significant difference.
Yet, can prolong storage time of fixing Niu Jingzi to the further optimization of described condition of storage and immobilization operation.The mensuration that the glucose consumption and the lactate generation of on-fixed sperm in fixing Niu Jingzi and the reference sample are carried out shows that the sperm metabolic rate that is stored in the calcium alginate gel network reduces (referring to embodiment 2).
Whether survive under physiological condition (being the activity of the described sperm condition should be maximum the time) for the evaluate fixed sperm, under 37 ℃ temperature, store experiment.The data show of the experiment of the Niu Jingzi of external storage and boar sperm is in Fig. 2 under next comfortable 37 ℃.
Viewed in the experiment as external storage at ambient temperature, described reference sample and the alginates fixedly motility rate between the sample of sperm are variant after described sample preparation.Yet in 37 ℃ of processes that store down, Niu Jingzi and the boar sperm motility rate in containing the reference sample of on-fixed sperm all reduces quite soon.In containing the sample of fixing sperm, the reduction of described motility rate is significantly slower.External storage is after 15 hours down at 37 ℃, and the motility rate of boar sperm in the pearl of two kinds of sizes is all still greater than 50%.In fact, storage is after 60 hours down at 37 ℃, and in the pearl of 3mm diameter, described motility rate still is about 30%.
As if in this experimental system, the motility rate of Niu Jingzi descends sooner than boar sperm.Yet, as using the boar sperm, in this experiment, also have significant difference between the fall off rate of Niu Jingzi motility rate in storage process.After storing 24 hours, fixing Niu Jingzi still has 45% motility rate, and does not record motility rate in described reference sample.
What need keep firmly in mind is, the data that present in Fig. 2 are to use artificial experimental system to obtain, and described system is not represented the internal milieu of jenny.Yet the data that present in Fig. 2 have shown really, Niu Jingzi and boar sperm in being fixed in the calcium alginate gel network in, even under physiological temp, also can survive and can in the long duration, keep motility rate.These data can also show, in order to keep the motility rate of sperm in the storage process under the physiological temp, can use the calcium alginate gel network to fix.This may be a particular importance, because the fertilization result in order to obtain, the time of insemination is very crucial.Because survival time of boar sperm is limited after insemination, so the insemination time is most important.Therefore, described result can show, in order to prolong the time period from insemination to fertilization, can use the sperm fixation also in conjunction with the controllable release after the insemination.
The insemination test
Carried out the insemination test in the swinerys of totally 9 sows at one.Directly begin to select sow, and select about 4 weeks that continue to after the insemination from the wean of last farrow.
In the slaughter house, after taking out internal organ, directly collect reproductive organs and in 15 minutes, check.The number of embryo's (if there is) in the number of corpus luteum and the uterine horn in calculating and the record ovary.By intervening, make that (even degeneration) embryo's of observing all existence possibility is higher relatively at this gestation commitment.The number of piggy by observing mature production thinks that it has the higher danger that loses the embryo of big percentage, and this is because the conceptus and the embryo of any death will be absorbed by the parent system again before ossified, finally can not see vestige.Nearly 30% fertilization pig ovum may dead and disappearance to skeletonization from becoming pregnant.In our test, can be by counting to such an extent that every sow embryo's during 4 weeks number and the number of corpus luteum calculate reliable fertilization rate estimated value.
The time of inseminating can normal time to than not waiting Zao one day of normal time, and carried out single and inseminated and twice insemination.The results are shown in the table 1 of these insemination tests.
Table 1: the number of insemination sow in the insemination test, pregnancy rate and (in the animal of gestation) fertilization rate.
| Method | The # sow | Pregnancy rate (%) | Fertilization rate (%) |
| Fixing sperm, the 1st 1+ 2 days | ?5 | ??4(80％) | ??57％ |
| Rong Xie fixedly sperm again, 1+2 days | ?4 | ??2(50％) | ??66％ |
1The 1st day=gather sperm same day
Also carried out the insemination test with Niu Jingzi.Owing to do not have a large amount of available laboratory animal, be not easy to quantitatively with these tests.In the cows that 20 available heifers are arranged, these experiments have been carried out.Before insemination with the synchronization in estrus of described heifer.Use fixing Niu Jingzi to obtain good fertilization rate.In described research, in 12 heifers with the insemination of 24 hours fixedly sperm of storage 6 fertilizations are arranged, this obtains conclusive evidence by the gestation contrast.
Described sperm is fixed in the alginates, at ambient temperature the different time period of stationary storage, dissolves the back or does not dissolve and inseminate.The time of inseminating normal time to than not waiting Zao one day of normal time, and only carried out the single insemination.5-7 week is confirmed animal pregnancy by rectal examination after insemination.The results are shown in the table 2 of these tests.
Table 2: the insemination test in ox: the number and the pregnancy rate of employed method, insemination heifer.
| The insemination test | Method | The # heifer | Pregnancy rate (%) |
| ??1 | Fixing sperm stores 24 hours down at 18 ℃, insemination immediately after the dissolving | 12 | ??6(50％) |
| ??2 | Fixing sperm stores 24 hours down at 18 ℃, with respect to estrus than normal Zao one day, do not dissolve and inseminate | 3 | ??1(33,3％) |
| ??3 | Fixing sperm stores 48 hours down at 18 ℃, insemination immediately after the dissolving | 5 | ??4(80％) |
These data show, the sperm that is fixed in the calcium alginate pearl not only can keep motility rate, but also the necessary all functions characteristic that can keep being fertilized.Therefore, Geno and Norsvin are verified, and the sperm that is fixed in the calcium alginate gel can be used for insemination, and can cause the fertilization of ox and pig.
Be regardless of and be limited to a kind of concrete theory, think that fixing sperm causes the minimizing of the energy that sperm consumes, this is useful for storage period and their fertility.
Therefore, in order to study this theory, with Niu Jingzi with about 150 * IO
6The concentration fixed of sperm/ml is in the alginates particle, and being added to 2 times of reference samples that in the newborn dilution buffer liquid of described pearl volume, prepared the sperm that contains free suspension from the homogeneous ejaculation, this reference sample contains identical total sperm concentration in each newborn dilution buffer liquid cumulative volume.Under 20 ℃, before the storage, at N
2The oxygen free condition of air-flow prepares described sample.In storage process, described newborn dilution buffer liquid is taken a sample, and measure the lactate that described sperm produces in described sample by HPLC.Described the results are shown in the table 3:
Table 3: the lactate that fixing sperm and the free sperm that suspends produce in the storage process at ambient temperature.Numerical value provides with the Lactated nanomole number that per 35,000 ten thousand sperms produce.
| Storage time (my god) | Fixing sperm | The reference sample |
| ??1 | ??4,21 | ??16,1 |
| ??2 | ??6,00 | ??20,6 |
| ??3 | ??6,90 | ??22,1 |
Embodiment 3: silver fox (silver fox) (rde fox (vulpes vulpes)) seminal fluid fixing
To in the EDTA thinner, be diluted among every ml from the mixed semen of the first qualities of 3 male adult silver foxes (rde fox) and contain 228 * 10
6Spermatid, and be transported in the laboratory.Gathering and diluting back 3 hours, with the seminal fluid of aliquot with the centrifugal 10min of 2000rpm.Sediment is mixed with alginates (LF 10/60), and described in present patent application, form pearl.Described pearl is stored among the Biladyl of improvement under 18 ℃.To remain seminal fluid is stored among the EDTA in contrast in 18 ℃.
After 48 hours, described pearl is dissolved.The seminal fluid of described stripping and contrast seminal fluid all are heated to 35 ℃ reach 20 minutes, then on the heating plate under 38 ℃ by phase contrast microscopy 10 * and 25 * multiplication factor under assess motility rate with the percentage form of motile sperm.
Table 4: stationary storage and motility rate revocable (contrast) silver fox seminal fluid
Embodiment 4: with piggy born after the normal gestation period behind the fixing boar semen single intrauterine insemination of alginates
Handled the mixed semen from 3 adult boars according to the present invention, and described pearl is used for the intrauterine insemination of sow, afterwards, sow is farrowing after full-term pregnancy.Preceding a brood of young wean on February 8th, 2007 with described sow, and in once inseminating with the seminal fluid that is fixed in the alginates pearl on February 12nd, 2007.Described sow gives birth to 16 viviparous piggys on June 10th, 2007.No stillbirth piggy in the described porkling, and all piggys are normal through clinical examination.
Embodiment 5: the ram seminal fluid is fixed in the alginates
With artificial vagina from two AI ram collecting semens and in 10 minutes, deliver to the laboratory.Before adding alginates, described seminal fluid is diluted in (Curtis PG in the thinner based on skimmed milk with 1+3, Forteath AD, Polge C.Survival of bull sperm frozen in milk diluentscontaining varying concentrations of glycerol and fructose.ProcIVth Int Congr Anim Reprod 1961; 3:952-956), and described in patent application 0613288.0 embodiment 1 form pearl.With described pearl down or be stored at ambient temperature in the thinners based on skimmed milk at 5 ℃.After fixing 24 hours and 48 hours, fixing sperm is discharged from described pearl, assess motility rate by microscope with the percentage of motile sperm cell then.Owing to real reason, in this research, do not comprise contrast.Yet, ram seminal fluid in liquid state descends in 30 hours the process of storage in 5 ℃ or 20 ℃, the motility parameters that comprises motility rate will variation (Paulenz H, Soderquist L, Perez-Pe R, Berg KA.Effect of differentextenders and storage temperatures on sperm viability of liquid ramsemen.Theriogenology 2002:57 (2): 823-36).
The fixing ram seminal fluid of table 5 stores the motility rate of 24 hours and 48 hours under different temperatures
Claims (34)
1. the biopolymerization composition granule that is used for the preservation sperm, wherein said sperm are embedded in the biopolymer gel network, and wherein the described biopolymer of the described sperm of embedding comprises the alginates that are rich in guluronic acid.
2. according to the particle of claim 1, wherein the described biopolymer of the described sperm of embedding comprises calcium alginate.
3. according to the particle of claim 1 or 2, wherein said biopolymer comprises low viscous alginates.
4. according to each particle of claim 1-3, wherein said concentration of alginate is at least 0.1%.
5. according to the particle of claim 4, wherein said concentration of alginate is at least 0.1% to 6% alginates.
6. according to the particle of claim 5, wherein said concentration of alginate is at least 1%.
7. according to the particle of claim 4, wherein said concentration of alginate is at least 2%.
8. according to the particle of claim 4, wherein said concentration of alginate is 6%.
9. according to each particle of claim 1-8, described particle has at least 0.1 * 10
6The sperm concentration of sperm/ml.
10. according to the particle of claim 9, described particle has at least 100 * 10
6The sperm concentration of sperm/ml.
11. according to the particle of claim 9, described particle has at least 2.5 * 10
9The sperm concentration of sperm/ml.
12. according to each particle of claim 1-11, described particle is stored in the solution.
13. according to the particle of claim 12, wherein said storage liquid: the biopolymer proportion of particles is at least 1: 1 to 1: 100.
14. according to the particle of claim 12-13, the embedding altogether of wherein said sperm and one or more antioxidant.
15. particle according to claim 14, wherein said antioxidant is selected from acetonate, 2,2,6,6-tetramethyl-piperidyl-1-oxygen, 4-hydroxyl-2,2,6,6-tetramethyl-piperidyl-1-oxygen, superoxide dismutase, catalase, glutathione peroxidase, Butylated Hydroxytoluene and Butylated Hydroxyanisole.
16. according to each particle of claim 1-15, wherein said particle is coated.
17. according to the particle of claim 16, wherein said dressing is selected from poly-D-lysine, shitosan, sulfate cellulose, hydroxypropyl methylcellulose and diallyl dimethyl ammoniumchloride.
18. according to each particle of claim 1-17, wherein said sperm also with to fertility and/or useful compound or the reagent embedding altogether of animal health.
19. according to the particle of claim 18, wherein said sperm and one or more are selected from following compound or reagent embedding altogether: thinner, cryoprotector, antibiotic, antibody, antioxidant, albumen and hormone.
20. according to each particle of claim 1-20, wherein said sperm cell source is from being selected from following animal: pig, ox, horse, sheep, goat, rabbit, poultry, for example pet, fleece animal, aquatic animal and the animals on the brink of extinction kind of purebred dog.
21. according to the particle of claim 20, wherein said sperm collection is to being selected from following animal: pig, ox, fleece animal and horse.
22. according to each particle of claim 1-21, wherein said sperm is included in the seminal fluid.
23. according to each particle of claim 1-22, wherein said particle is further handled through dehydration, cryopreservation or freeze drying.
24. according to each particle of claim 1-22, wherein said particle directly forms in the seminal fluid container.
25. be used to prepare each the method for particle according to claim 1-24, wherein the mixture with alginates and sperm is added in the gelling soln.
26. according to the method for claim 25, wherein said gelling soln comprises one or more and is selected from following ion: calcium, sodium, barium and magnesium.
27. according to the method for claim 26, wherein said gelling soln comprises calcium ion and sodium ion.
28. according to each method of claim 25-27, wherein said particle is further handled through dehydration, cryopreservation or freeze drying.
29. according to each the purposes of biopolymerization composition granule in animal breeding of claim 1-24.
30. according to the purposes of claim 29, wherein said particle is used to be selected from the breeding of following animal: pig, ox, horse, sheep, goat, rabbit, poultry, for example pet, fleece animal, aquatic animal and the animals on the brink of extinction kind of purebred dog.
31. according to the purposes of claim 29 or 30, wherein said particle is directly inseminated.
32., wherein before insemination, dissolve described particle according to the purposes of claim 29 or 30.
33. according to the purposes of claim 29 or 30, wherein said particle uses with free sperm.
34. the method for animal fertilization wherein will be preserved in according in the importing of the sperm in the particle of the claim 1-24 receptor jenny.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0613288.0 | 2006-07-04 | ||
| GB0613288A GB0613288D0 (en) | 2006-07-04 | 2006-07-04 | Preservation and controlled delivery/release of spermatoza |
| US80771606P | 2006-07-19 | 2006-07-19 | |
| US60/807,716 | 2006-07-19 | ||
| PCT/NO2007/000256 WO2008004890A2 (en) | 2006-07-04 | 2007-07-03 | Preservation and controlled delivery/release of spermatozoa |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101616578A true CN101616578A (en) | 2009-12-30 |
| CN101616578B CN101616578B (en) | 2015-08-12 |
Family
ID=36926446
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200780025213.8A Active CN101616578B (en) | 2006-07-04 | 2007-07-03 | Preservation and controlled the sending of sperm are passed/are discharged |
Country Status (11)
| Country | Link |
|---|---|
| CN (1) | CN101616578B (en) |
| BR (1) | BRPI0713899A2 (en) |
| DK (1) | DK2048943T3 (en) |
| ES (1) | ES2525327T3 (en) |
| GB (1) | GB0613288D0 (en) |
| NZ (1) | NZ574510A (en) |
| PT (1) | PT2048943E (en) |
| RU (1) | RU2438309C2 (en) |
| SI (1) | SI2048943T1 (en) |
| UA (1) | UA98467C2 (en) |
| ZA (1) | ZA200900641B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107251892A (en) * | 2017-05-25 | 2017-10-17 | 中国热带农业科学院热带作物品种资源研究所 | It is a kind of suitable for the solution for dilution of cock semen of torrid areas, preparation and its application process |
| CN110035656A (en) * | 2016-12-05 | 2019-07-19 | 斯博姆维透公司 | Slow releasing composition |
| CN112367841A (en) * | 2018-06-04 | 2021-02-12 | 斯博姆维透公司 | Functionalized kit for preparing hydrogel |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2504581C2 (en) * | 2012-02-02 | 2014-01-20 | Константин Владимирович Кириенко | Method for vital immobilisation of human and animal sperm cells |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5876742A (en) * | 1994-01-24 | 1999-03-02 | The Regents Of The University Of California | Biological tissue transplant coated with stabilized multilayer alginate coating suitable for transplantation and method of preparation thereof |
-
2006
- 2006-07-04 GB GB0613288A patent/GB0613288D0/en not_active Ceased
-
2007
- 2007-03-07 UA UAA200900820A patent/UA98467C2/en unknown
- 2007-07-03 SI SI200731558T patent/SI2048943T1/en unknown
- 2007-07-03 PT PT07793911T patent/PT2048943E/en unknown
- 2007-07-03 BR BRPI0713899-7A patent/BRPI0713899A2/en active Search and Examination
- 2007-07-03 ES ES07793911.4T patent/ES2525327T3/en active Active
- 2007-07-03 ZA ZA200900641A patent/ZA200900641B/en unknown
- 2007-07-03 RU RU2009103200/21A patent/RU2438309C2/en active
- 2007-07-03 NZ NZ574510A patent/NZ574510A/en unknown
- 2007-07-03 DK DK07793911.4T patent/DK2048943T3/en active
- 2007-07-03 CN CN200780025213.8A patent/CN101616578B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110035656A (en) * | 2016-12-05 | 2019-07-19 | 斯博姆维透公司 | Slow releasing composition |
| CN107251892A (en) * | 2017-05-25 | 2017-10-17 | 中国热带农业科学院热带作物品种资源研究所 | It is a kind of suitable for the solution for dilution of cock semen of torrid areas, preparation and its application process |
| CN112367841A (en) * | 2018-06-04 | 2021-02-12 | 斯博姆维透公司 | Functionalized kit for preparing hydrogel |
| CN112367841B (en) * | 2018-06-04 | 2023-06-09 | 斯博姆维透公司 | Functional kit for preparing hydrogel |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2438309C2 (en) | 2012-01-10 |
| ES2525327T3 (en) | 2014-12-22 |
| PT2048943E (en) | 2014-12-03 |
| ZA200900641B (en) | 2010-07-28 |
| DK2048943T3 (en) | 2014-12-15 |
| CN101616578B (en) | 2015-08-12 |
| NZ574510A (en) | 2011-11-25 |
| GB0613288D0 (en) | 2006-08-16 |
| BRPI0713899A2 (en) | 2012-12-11 |
| RU2009103200A (en) | 2010-08-10 |
| SI2048943T1 (en) | 2015-01-30 |
| UA98467C2 (en) | 2012-05-25 |
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