CN101014700A - Sperm suspensions for sorting into x or y chromosome-bearing enriched populations - Google Patents

Abstract

Sperm cell suspensions comprising a motility inhibitor are disclosed. The cells contained in such suspensions tend to have a greater capacity for enduring the various process steps typically associated with the sorting of sperm cells into gender enriched populations, thereby resulting in post-sort compositions with an increased number of viable or motile sperm. Processes for forming such cell suspensions, as well as processes for staining sperm cells, are also disclosed.

Description

Be used for the sperm suspensions that sorting has X or Y chromosome enriched populations

Invention field

The present invention relates generally to the method for sorting sperms cell.More specifically, the suspension that the present invention relates to prepare the spermatid mobility and reduce with respect to the sperm of ejaculating inside the body more specifically is that mobility temporarily reduces; Described suspension can be used for as the spermatid branch being elected as the method for the enriched sperm cell mass that has X or Y chromosome.

Background of invention

By artificial insemination (AI) make animal fertilization and in vitro fertilization after embryo transfer be a proven technique.In the animal industry of being in, can exert one's influence and make its offspring have one or more required features, remarkable advantages is arranged the breeding result.For example, if can select the offspring to help female in the milk-product industry in advance and guarantee the output of milk cow, then can bring economic benefit.Sperm is separated into the cell mass that has X and Y chromosome of enrichment, and promptly so-called gender enriched seminal fluid or gender enriched sperm are one of methods that reaches offspring's preliminary election purpose.

For obtaining the gender enriched seminal fluid, must divide then and elect the cell that has X and Y chromosome as with spermatid with dyeing.Each step of dyeing and assorting room for spermatid all be stress, can cause the viability or the mobility of spermatid, particularly before the reduction of tropism's mobility.

People such as Salisbury have described the bull ejaculum directly have been collected into technology in the diluent, and the mobility that described diluent is capable of inhibiting cell prevents that also it is from absorbing sugar the refining on every side.Penetrate seminal fluid to diluent when collecting, and pass through the CO of inflation 100% ₂When replacing the gas phase of liquid top, the cell that penetrates in the seminal fluid can lost-motion.As long as reside in this diluent cell and excluding air, just can guarantee its at room temperature a few hours do not move, and in the time of 5 ℃, can keep at least 8 days.

Summary of the invention

One of many-side of the present invention relates to sperm suspensions, and described sperm suspensions can be used for the method as the enriched sperm group who the sperm branch is elected as band X or Y chromosome.

Therefore in brief, the present invention relates to contain the spermatid suspension of the composition of sperm alive and downward modulation sperm Sugar intake, the sperm concentration in the described suspension is less than about 1 * 10 ⁶Sperm/ml or be at least 1 * 10 ⁸Sperm/ml.

The invention still further relates to the spermatid suspension that contains the sperm alive that do not move, the sperm concentration in the described suspension is less than about 1 * 10 ⁶Sperm/ml or be at least 1 * 10 ⁸Sperm/ml.

The invention still further relates to the spermatid suspension that contains sperm alive, described sperm has more the mobility characteristics of sperm in the epididymis with respect to the sperm that penetrates in the same germline body, and the sperm concentration in the described suspension is less than about 1 * 10 ⁶Sperm/ml or be at least 1 * 10 ⁸Sperm/ml.

The invention still further relates to and contain sperm alive, potassium, and randomly contain the spermatid suspension of sodium, the sperm concentration in the described suspension is at least 1 * 10 ⁸Sperm/ml, and the mol ratio of potassium and sodium was greater than each 1: 1.

The invention still further relates to the composition that contains sperm alive, downward modulation sperm Sugar intake and the spermatid suspension of DNA selective dye.

The invention still further relates to the spermatid suspension that contains do not move sperm alive and DNA selective dye.

The invention still further relates to the spermatid suspension that contains sperm alive and DNA selective dye, described sperm is with respect to the sperm that penetrates in the same germline body, and its metabolic rate and mobility have more the characteristics of sperm in the epididymis.

The invention still further relates to the spermatid suspension that contains the sperm alive that do not move, be combined with the DNA selective dye on the DNA of described sperm.

The invention still further relates to the spermatid suspension that contains sperm alive, for the sperm that penetrates in the same germline body, described sperm metabolic rate and mobility have more the characteristics of sperm in the epididymis, and also are combined with the DNA selective dye on its DNA.

The invention still further relates to the painted method of spermatid, described method comprises the dye mixture that formation is such, and it contains the potassium of complete spermatid alive, motion inhibition amount, and the DNA selective dye.

The invention still further relates to the method for the spermatid suspension that is formed for the flow cytometry method, described method comprises mixes the spermatid source with the composition that suppresses the spermatid mobility, to form spermatid suspension, spermatid concentration is less than about 1 * 10 in the described suspension ⁶Spermatid/ml or be at least 1 * 10 ⁸Spermatid/ml.

The invention still further relates to the method for the spermatid suspension that is formed for the flow cytometry method, described method comprise with Mammals penetrate seminal fluid collecting to the damping fluid of the depressomotor that contains the inhibition amount to form sperm suspensions, contain sperm concentration in the described suspension less than about 1 * 10 ⁶Spermatid/ml or be at least 1 * 10 ⁸Spermatid/ml.

Others of the present invention and feature are conspicuous, and part will be in hereinafter explanation.

The accompanying drawing summary

Fig. 1 is with the pictorialization result who studies among the embodiment 1, wherein measured in the TCA that contains the 10mM pyruvate salt of 28 ℃ of TCA that contain the 10mM pyruvate salt or carbonic acid gas gas-bearing formation parcel, with the preceding tropic movement per-cent of the spermatid of 600 μ M Hoechst, 33342 dyeings.

Fig. 2 is with the pictorialization result who studies among the embodiment 1, wherein measured in 28 ℃ of TCA or the inhibitor of pH7.3 that contain the 10mM pyruvate salt, with the preceding tropic movement per-cent of the spermatid of 600 μ MHoechst, 33342 dyeings based on carbonate.

Fig. 3 is with the pictorialization result who studies among the embodiment 1, wherein measured in 28 ℃ of TCA or the inhibitor of pH6.2 that contain the 10mM pyruvate salt, with the preceding tropic movement per-cent of the spermatid of 600 μ MHoechst, 33342 dyeings based on carbonate.

Fig. 4 is with the pictorialization result who studies among the embodiment 2, wherein measured in 28 ℃ and contained the TCA of 10mM pyruvate salt and, with the preceding tropic movement per-cent of the spermatid of 1000 μ M Hoechst, 33342 dyeings subsequently with containing under the situation that the TCA of 10mM pyruvate salt or the pH6.2 inhibitor 1 to 3 based on carbonate dilutes.

Fig. 5 is with the pictorialization result who studies among the embodiment 2, wherein measured in 28 ℃ (1) contain the TCA of 10mM pyruvate salt and with the situation of same solution 1 to 3 dilution under or (2) pH7.3 based on the damping fluid of carbonate and the situation of diluting based on the inhibitor 1 to 3 of carbonate with pH6.2 under, with the preceding tropic movement per-cent of the spermatid of 1000 μ M Hoechst, 33342 dyeings.

Fig. 6 is with the pictorialization result who studies among the embodiment 2, wherein measured in 28 ℃ of TCA or the inhibitor of pH6.2 that contain the 10mM pyruvate salt, with the preceding tropic movement per-cent of the spermatid of 1000 μ MHoechst, 33342 dyeings based on carbonate.

Fig. 7 is with the pictorialization result who studies among the embodiment 3, wherein measured in 41 ℃ and contained the TCA of 10mM pyruvate salt and, with the preceding tropic movement per-cent of the spermatid of 300 μ M Hoechst, 33342 dyeings subsequently with containing under the situation that the TCA of 10mM pyruvate salt or the pH6.2 inhibitor 1 to 3 based on carbonate dilutes.

Fig. 8 is with the pictorialization result who studies among the embodiment 3, wherein measured in 41 ℃ (1) contain the TCA of 10mM pyruvate salt and with the situation of same solution 1 to 3 dilution under or (2) pH7.3 based on the damping fluid of carbonate and the situation of diluting based on the inhibitor 1 to 3 of carbonate with pH6.2 under, with the preceding tropic movement per-cent of the spermatid of 300 μ M Hoechst, 33342 dyeings.

Fig. 9 is with the pictorialization result who studies among the embodiment 3, wherein measured in 41 ℃ of TCA or the inhibitor of pH6.2 that contain the 10mM pyruvate salt, with the preceding tropic movement per-cent of the spermatid of 300 μ MHoechst, 33342 dyeings based on carbonate.

Figure 10 is with the pictorialization result who studies among the embodiment 4, wherein measured in 41 ℃ of TCA damping fluids or contains in the TCA damping fluid of 10mM pyruvate salt, with the preceding tropic movement per-cent of the sperm of 400 μ M Hoechst33342 dyeings.

Figure 11 is with the pictorialization result who studies among the embodiment 5, wherein measured in 41 ℃ of TCA damping fluids or contains in the TCA damping fluid of 10 μ M vitamin Ks, with the preceding tropic movement per-cent of the sperm of 400 μ M Hoechst33342 dyeings.

Figure 12 is with the pictorialization result who studies among the embodiment 6, wherein measured in 41 ℃ of TCA damping fluids or contains in the TCA damping fluid of 100 μ M vitamin Ks, with the preceding tropic movement per-cent of the sperm of 400 μ M Hoechst33342 dyeings.

Figure 13 is with the pictorialization result who studies among the embodiment 7, wherein measured in 41 ℃ of TCA damping fluids or contains in the TCA damping fluid of 1mM Thioctic Acid, with the preceding tropic movement per-cent of the sperm of 400 μ M Hoechst, 33342 dyeings.

Figure 14 is with the pictorialization result who studies among the embodiment 8, wherein measured in 28 ℃ of TCA damping fluids or contains in the TCA damping fluid of 10mM pyruvate salt, with the preceding tropic movement per-cent of the sperm of 600 μ M Hoechst33342 dyeings.

Figure 15 is with the pictorialization result who studies among the embodiment 9, wherein measured in 28 ℃ of TCA damping fluids or contains in the TCA damping fluid of 100 μ M vitamin Ks, with the preceding tropic movement per-cent of the sperm of 600 μ M Hoechst33342 dyeings.

Figure 16 is with the pictorialization result who studies among the embodiment 10, wherein measured in 28 ℃ of TCA damping fluids or contains in the TCA damping fluid of 1mM Thioctic Acid, with the preceding tropic movement per-cent of the sperm of 600 μ M Hoechst, 33342 dyeings.

Figure 17 is with the pictorialization result who studies among the embodiment 11, wherein measured in 28 ℃ of TCA damping fluids, contain the 2.5mM pyruvate salt the TCA damping fluid, contain the 10mM pyruvate salt the TCA damping fluid, contain the TCA damping fluid of 25mM pyruvate salt and contain in the TCA damping fluid of 50mM pyruvate salt, with the preceding tropic movement per-cent of the sperm of 600 μ M Hoechst, 33342 dyeings.

Figure 18 is with the pictorialization result who studies among the embodiment 12, wherein measured in 28 ℃ of TCA damping fluids or contains in the TCA damping fluid of 10mM pyruvate salt, with the preceding tropic movement per-cent of the sperm of 20 μ M SYBR-14 dyeings.

Figure 19 is with the pictorialization result who studies among the embodiment 13, wherein measured in 28 ℃ of TCA damping fluids or contains in the TCA damping fluid of 10mM pyruvate salt, with the preceding tropic movement per-cent of the sperm of 100 μ M BBC dyeings.

Figure 20 is with the pictorialization result who studies among the embodiment 14, wherein measured in 28 ℃ of TCA damping fluids or contains in the TCA damping fluid of 10mM pyruvate salt, with the preceding tropic movement per-cent of the sperm of 200 μ M BBC dyeings.

Detailed Description Of The Invention

Surprisingly, determined the sperm with respect to the reduction of the sperm of ejaculating inside the body (system mutually of the same race) motility, often more can tolerate a plurality of is with the common relevant step of the sperm enriched populations of X or Y chromosome with the sorting sperms cell. Therefore in preferred embodiments, can be for the preparation of test-tube gender enriched sperm group, the living cells number increases or motion sperm (particularly propulsion sperm) number increases in the composition of described sperm group after dyeing or after the sorting.

The method according to this invention, formation contains the suspension of the composition of sperm and one or more Inhibit sperm motilities, sometimes also is called dispersion; The suppressed state of this kind motility sometimes be referred to as do not move or sperm static. Generally speaking, the density that contains sperm in the suspension is about 1 * 10³Sperm/ml is to about 5 * 10¹⁰Sperm/ml suspension. For example, in one embodiment, can contain the sperm of " relatively low " density in the suspension, namely sperm concentration is less than about 1 * 10⁷Sperm/ml preferably is less than about 1 * 10⁶Sperm/ml, more preferably from about 1 * 10³To about 5 * 10⁶Sperm/ml, more preferably from about 1 * 10³To about 1 * 10⁶Sperm/ml, even more preferably 1 * 10⁴To about 1 * 10⁵Sperm/ml, most preferably from about 1 * 10⁵Sperm/ml suspension. In optional embodiment, can contain the sperm of " centre " density in the suspension, namely density is about 1 * 10⁷To about 1 * 10⁸Sperm/ml suspension. And in another embodiment, can contain the sperm of " relatively high " density in the suspension, namely sperm concentration is at least about 1 * 10⁸Sperm/ml, preferred about 1 * 10⁸To about 5 * 10¹⁰Sperm/ml, more preferably from about 1.5 * 10⁸To about 2 * 10¹⁰Sperm/ml, even more preferably 1.5 * 10⁸To about 2 * 10⁸Sperm/ml is more preferably about 1.5 * 10⁸Sperm/ml suspension. Therefore for example, in one embodiment, can contain at least about 1.25 * 10 in the suspension⁸, at least about 1.5 * 10⁸, at least about 1.75 * 10⁸, at least about 2 * 10⁸, at least about 2.25 * 10⁸, at least about 2.5 * 10⁸, at least about 2.75 * 10⁸, or even at least about 3 * 10⁸Sperm/ml suspension. In optional embodiment, can contain in the suspension and be less than about 9 * 10⁵, be less than about 7 * 10⁵, be less than about 5 * 10⁵, be less than about 2 * 10⁵, be less than about 1 * 10⁵, be less than about 1 * 10⁴, or even less than about 1 * 10³Sperm/ml suspension.

Sperm concentration in the sperm suspensions depends on some considerations, and comprising subsequently will be for the method for enrichment or sorting sperms cell. For example, can utilize flow cytometry sorting sperms cell as will be detailed later. Under these circumstances, the buffering sperm suspensions generally contains the sperm of " centre " or " relatively high " density. Other sorting or beneficiation technologies may be more conducive to use when sperm concentration is low, for example indicate the sperm of " relative low " density of mark (as described here dyestuff and mark).

In preferred embodiments, the sperm in the suspension of the present invention shows the characteristic behavior of sperm in the epididymis in some aspects; For example, the not motility of sperm, and/or for washing or the sperm that just penetrated, its internal respiration speed is lower and speed aerobic glycolysis is higher. Best suppressed sperm is with after inhibitor separates, and its motility can show the characteristic behavior (and not having the characteristics of epididymal sperm) of ejaculated sperm; And in one embodiment, its motility and breathing all can have the characteristic performance such as ejaculated sperm.

For example in one embodiment, analyze (Hamilton-Thorne HTM-IVOS area of computer aided sperm analysis system by the HTM-IVOS sperm, Hamilton-Thorne Research, Beverly MA) measures, depressomotor can make the path velocity of spermatoblast in the dispersion, front tropism's speed or both whiles just penetrate the path velocity of spermatoblast in the seminal fluid, front tropism's speed or both whiles with respect to system mutually of the same race, reduces at least about 50%. Preferably by HTM-IVOS sperm analysis to measure, depressomotor can make the path velocity of spermatoblast in the dispersion, front tropism's speed or both whiles just penetrate the path velocity of spermatoblast in the seminal fluid, front tropism's speed or both whiles with respect to system mutually of the same race, reduces at least about 60%. More preferably by HTM-IVOS sperm analysis to measure, depressomotor can make the path velocity of spermatoblast in the dispersion, front tropism's speed or both whiles just penetrate the path velocity of spermatoblast in the seminal fluid, front tropism's speed or both whiles with respect to system mutually of the same race, reduces at least about 70%. Also more preferably by HTM-IVOS sperm analysis to measure, depressomotor can make the path velocity of spermatoblast in the dispersion, front tropism's speed or both whiles just penetrate the path velocity of spermatoblast in the seminal fluid, front tropism's speed or both whiles with respect to system mutually of the same race, reduces at least about 80%. Even more preferably by HTM-IVOS sperm analysis to measure, depressomotor can make the path velocity of spermatoblast in the dispersion, front tropism's speed or both whiles just penetrate the path velocity of spermatoblast in the seminal fluid, front tropism's speed or both whiles with respect to system mutually of the same race, reduces at least about 90%. Even more preferably by HTM-IVOS sperm analysis to measure, depressomotor can make the path velocity of spermatoblast in the dispersion, front tropism's speed or both whiles just penetrate the path velocity of spermatoblast in the seminal fluid, front tropism's speed or both whiles with respect to system mutually of the same race, reduces at least about 95%. Most preferably by HTM-IVOS sperm analysis to measure, depressomotor can make the path velocity of spermatoblast in the dispersion, front tropism's speed or both whiles just penetrate the path velocity of spermatoblast in the seminal fluid, front tropism's speed or both whiles with respect to system mutually of the same race, reduces at least about 99%.

Except the inhibition buffer solution or alternative be also can reduce separately the temperature of spermatoblast or the environment temperature of next-door neighbour's spermatoblast (being sperm dispersion system) to affect the motility of cell. This kind cooling generally can increase not motility. In addition, for example, spermatoblast or sperm dispersion are that the reduction of temperature can cause be used to inducing the not concentration reduction of the inhibitor of motility. Therefore, sperm dispersion system can be in and be no more than 5 ℃ temperature; Preferably between about 0 ℃ to about 5 ℃; More preferably between about 3 ℃ to about 5 ℃; And most preferably be about 5 ℃. Perhaps the temperature of sperm dispersion system can between about 4 ℃ to about 50 ℃ of scopes; Be preferably about 7 ℃ to about 43 ℃; More preferably about 10 ℃ to about 39 ℃; Also more preferably about 15 ℃ to about 30 ℃; Even more preferably about 17 ℃ to about 25 ℃; And most preferably be about 18 ℃. Yet, preferably spermatoblast is not exposed to and can causes under the dysgenic temperature of essence cell viability.

Inhibitor can be a series of in the inhibited composition of sperm motility any one. Such composition comprises such as sodium/potassium ATP enzyme inhibitor, such as unabain; The composition that contains potassium ion; And the composition that contains potassium ion and sodium ion. For example, the potassium ion of high concentration can the Inhibit sperm motility relatively in the suspension. Therefore generally speaking, the potassium concn that contains in potassium ion source and the suspension in the preferred suspension is at least about 0.05mol/L. More preferably potassium concn is at least about 0.05mol/L to about 0.5mol/L. Also more preferably potassium concn is at least about 0.1mol/L to about 0.3mol/L. Most preferably potassium concn is about 0.173 mol/L. This type of suspension general (but nonessential) also contains source of sodium ions. When containing sodium ion, the mol ratio of potassium and sodium generally is equal to or greater than each 1: 1. The mol ratio of preferred potassium and sodium is at least about 1.25: 1. More preferably the mol ratio of potassium and sodium is at least about 1.5: 1. Also more preferably the mol ratio of potassium and sodium is at least about 1.75: 1. Also more preferably the mol ratio of potassium and sodium is at least about 1.78: 1. In a specific embodiment, the mol ratio of potassium and sodium is at least about 2: 1. And in another embodiment, the mol ratio of potassium and sodium is at least about 3: 1. Again in another embodiment, the mol ratio of potassium and sodium is at least about 4: 1. Again in another embodiment, the mol ratio of potassium and sodium is at least about 5: 1. Again in another embodiment, the mol ratio of potassium and sodium is at least about 6: 1. Again in another embodiment, the mol ratio of potassium and sodium is at least about 7: 1. Again in another embodiment, the mol ratio of potassium and sodium is at least about 8: 1.

Also can additionally contain ion or the carbon dioxide source that to reduce Sugar intake in the sperm suspensions. In this embodiment, the source of carbon dioxide can be for such as one or more carbonate. In a present preferred embodiment, sperm suspensions contains NaHCO₃And KHCO₃, potassium and source of sodium ions and partial pressure of carbon dioxide are provided thus. For example, in a present preferred embodiment, sperm suspensions contains NaHCO₃And KHCO₃Aqueous solution, preferred NaHCO₃、KHCO ₃And C₆H ₈O ₇·H ₂The aqueous solution of O; Generally speaking, KHCO in the dispersion₃Concentration can be at least about 0.05mol/L. More preferably KHCO₃Concentration be at least about 0.05mol/L to about 0.5mol/L. More preferably KHCO also₃Concentration be at least about 0.1mol/L to about 0.3mol/L. In particularly preferred embodiments, such as Salisbury ﹠ Graves, J.Reprod.Fertil, disclosed such among the 6:351-359 (1963), utilize to contain 0.097mol/L NaHCO₃、0.173mol/L KHCO ₃、0.090 mol/L C ₆H ₈O ₇·H ₂The inhibition buffer solution of the O aqueous solution forms suspension. As long as spermatoblast is exposed to depressomotor, they generally all can keep static.

In dispersion, contain C₆H ₈O ₇·H ₂During O, KHCO₃With NaHCO₃Between mol ratio can be as mentioned above. KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O generally is equal to or greater than each 1: 1, but generally can not surpass mol ratio 8: 1. Preferred KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 1.25: 1. More preferably KHCO also₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 1.5: 1. More preferably KHCO also₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 1.75: 1. In a specific embodiments, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 1.78: 1. In another embodiment, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 2: 1. And in another embodiment, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 3: 1. Again in another embodiment, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 4: 1. Again in another embodiment, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 5: 1. Again in another embodiment, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 6: 1. Again in another embodiment, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 7: 1. Again in another embodiment, KHCO₃With C₆H ₈O ₇·H ₂Mol ratio between O is at least about 8: 1. In particularly preferred embodiments, such as Salisbury ﹠ Graves, J.Reprod.Fertil, disclosed such among the 6:351-359 (1963), utilize to contain 0.097mol/L NaHCO₃、0.173mol/L KHCO ₃、 0.090mol/L C ₆H ₈O ₇·H ₂The inhibition buffer solution of the aqueous solution of O forms suspension. As long as spermatoblast is exposed to depressomotor, they generally all can keep static.

Experimental evidence is up to now also pointed out, if spermatoblast suspension is kept under the atmosphere that increases with respect to the air partial pressure of carbon dioxide, can improve holistic health and other vital signs of spermatoblast. In preferred embodiments, the partial pressure of carbon dioxide value is at least 0.9 in the atmosphere of suspension top; More preferably at least about 0.95.

Cell can be separated with the motility inhibitor and be exposed in the air, make akinete be reduced to activated state. In addition, can also be by in physiological saline people such as (, 1963) Salisbury or come the initial activity state such as diluting cells in the buffer solution of TCA buffer solution or PBS. Generally speaking, according to HTM-IVOS sperm analysis to measure, at least about 20%, preferably at least about 50%, more preferably at least about 60%, also more preferably at least about 70% even more preferably at least about 80% even more preferably at least about 90%, also more preferably most preferably have such path velocity, front tropism's speed genetic system at least about 99% cell (being the reactivation cell) of replying activated state at least about 95%: namely be respectively with the motility inhibitor mixed before the path velocity, front tropism's speed of spermatoblast (namely just the spermatoblast of ejaculation) or both at least about 50%, preferably at least about 60%, more preferably at least about 70%, also more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 99%.

Generally speaking, cell sorting method comprises a series of separating steps, i.e. the collection of the collection of cell sample, cell dyeing, cell sorting, sorting cells, and randomly comprise the freezing stretching, extension (cryoextension) of sorting cells. Preferably comprise the motility inhibitor in the sperm suspensions that forms, or in one or more these steps, adopt it.

The collection of cell sample

Can collect the complete spermatoblast alive of ox, pig, horse or other mammal also contacts it with the motility inhibitor. The method of known numerous collection sperm alive for example, comprises hand grip, uses artificial vagina and electro-ejaculation. For example, can be with general every milliliter ox seminal fluid sample that contains 500,000,000 to 10,000,000,000 spermatoblasts, directly origin source mammal is collected in the container that contains the motility inhibitor, to form sperm suspensions. Alternatively, also sample of sperm can be collected in the empty, in several minutes to a few hours that collect, it be contacted to form sperm suspensions with the motility inhibitor subsequently.

Except that cushion, also can contain a series of additives in the sperm suspensions to strengthen sperm viability.Exemplary additive comprises protein source, microbiotic and regulates in the cell and/or the composition of extracellular oxidation/reduction reaction.

Exemplary protein source comprises yolk, yolk extract, breast (comprising hot homogenate and skimming milk), newborn extract, soybean protein, soybean protein extract, serum albumin, bovine serum albumin, human serum surrogate enriching substance and their combination.Albumin is bovine serum albumin (BSA) particularly, is preferred protein source.For example, if contain BSA, then the BSA content in the sperm suspensions can be less than about 5.0% (w/v), preferably is less than about 2% (w/v), and more preferably less than about 1% (w/v), and most preferably its amount is about 0.1% (w/v).

Use separately protein source (as BSA), may initial suspension in the capacitation process of one of percentage spermatid.This process preferably betides in the female reproductive tract.Therefore, in order to suppress the initial of capacitation during dilution and dyeing subsequently and the sorting, can contain alternative protein source or protein surrogate in the sperm suspensions.Described alternative protein source or protein surrogate also have the advantage of typical protein source (as BSA) except suppressing the initial capacitation of the cell of bigger per-cent in the sperm suspensions.The example in alternative white matter source comprises human serum surrogate enriching substance (SSS) (IrvineScientific, Santa Ana, CA) and the BSA that strengthens of cholesterol, and the example of protein surrogate comprises polyvinyl alcohol, for example general molecular weight is about 30,000 to about 60,000 be low to moderate the medium viscosity polyvinyl alcohol.Generally speaking, if contain these compositions, its content is identical with the amount of above-mentioned BSA; Total albumin content generally is no more than about 5.0% (w/v) in cushion or the buffered soln.

For example, regulate cell exemplary composition interior and/or the extracellular oxidation/reduction reaction and comprise pyruvate salt, vitamin K, Thioctic Acid, gsh, flavine, quinones, superoxide-dismutase (SOD) and SOD stand-in.If contain this based composition in the sperm suspensions, then its concentration is enough to produce protective effect and can produce detrimentally affect to sperm health.Exemplary concentration range comprises from about 10 μ M to about 20mM, depends on such as used concrete composition or the factors such as sperm concentration in the suspension.For example, the concentration of pyruvate salt can be about 1mM to about 20mM in the sperm suspensions, is preferably about 5mM to about 15mM, more preferably about 10mM.The concentration of vitamin K can be about 1 μ M to about 100 μ M in the sperm suspensions, is preferably about 10 μ M to about 100 μ M, more preferably about 100 μ M.The concentration of Thioctic Acid can be about 0.1mM to about 1mM in the sperm suspensions, is preferably about 0.5mM to about 1mM, and more preferably about 1mM.

Can contain microbiotic in the sperm suspensions to suppress the growth of bacterium.Exemplary microbiotic comprises for example tylosin, gentamicin, lincomycin, spectinomycin, sharp proceomycin Linco-Spectin ^(lincomycin hydroxy chlorination thing-spectinomycin), penicillin, Streptomycin sulphate, ticarcillin (ticarcillin) or its arbitrary combination.If contain microbiotic, then its concentration can be in every ml seminal fluid about 50 μ g to about 800 μ g (no matter seminal fluid is purified, resiliency or contains other material, any additive of for example mentioning) herein.Certified Semen Services (CSS) has issued the outline that relevant microbiotic is used with American National improvement of breed association (NAAB) in sperm is collected and used.

Can in sperm dispersion system, add somatomedin, to assist to keep the viability of spermatid.Exemplary somatomedin comprises as transforming growth factor (" TGF ") (for example TGF β-1 and TGF β-2), and rhIGF-1 (" IGF ") (for example IGF-1).Generally speaking, the form that the TGF in the sperm dispersion system can TGF β-1 occurs, its concentration for about 0.1ng/L to about 10 μ g/L, or be the form of TGF β-2, its concentration is extremely about 200ng/L of about 0.1ng/L; And IGF can be the IGF-1 form in sperm dispersion system, and its concentration is that about 0.1ng/L is to about 50 μ g/L.The purposes of this type of somatomedin is well known in the art, and is disclosed in as in the U.S. Patent Application Publication No. 2003/0157473, and its content is quoted as a reference herein.

In case collect cell, it can be preserved under a few hours, the low temperature (as 5 ℃) in stationary state under the room temperature and preserve several weeks, or preserve the several months in the freezing stretching, extension agent of being discussed hereinafter.The atmosphere of preferred cell top contains higher partial pressure of carbon dioxide as mentioned above.Perhaps, the cell of collection also can be used in a few hours as breeding method, dyeing process or sorting method.

Cell dyeing

The mobility inhibitor can be used for making cell not move during cell dyeing.Generally speaking, can comprise in the spermatid dyeing process forming dye mixture (being referred to as the mark mixture sometimes), wherein contain complete spermatid alive, mobility inhibitor and dyestuff (being referred to as mark sometimes).In this one side of the present invention, the mobility inhibitor can be contacted with spermatid to form sperm suspensions, then sperm suspensions is contacted with the DNA selective dye.In this embodiment, sperm source can be pure sperm, perhaps for by the semen derivative that contains sperm centrifugal or that use other means separated sperm fraction to obtain.

In optional embodiment, dyestuff can mix mutually with the mobility inhibitor, thereby forms dye solution.Therefore, for example, the dyestuff of pure solid (comprising free flowable powder) or forms of liquid compositions can be combined to form dye solution with inhibitor, and described solution can be subsequently mixes with pure sperm, sperm suspensions or the semen derivative that contains sperm.

In any case, as long as spermatid is kept in the inhibitor, generally all can keep static people such as (, 1963) Salisbury.Yet, preferably dye mixture is kept in the atmosphere that partial pressure of carbon dioxide increases for air; For example, the CO that contains 99%+ in the atmosphere above the general preferred dye mixture ₂

The pH of dye mixture can be remained in the certain pH value scope more arbitrarily; Generally speaking, this scope is about 5.0 to about 9.0.For example, can make dye mixture remain under " subacidity " pH promptly about 5.0 to about 7.0.In this embodiment, preferred pH is about 6.0 to about 7.0, more preferably about 6.0 to about 6.5, and most preferably is about 6.2.Perhaps, also can make dye mixture remain under " slight alkalinity " pH promptly about 7.0 to about 9.0.In this embodiment, preferred pH is about 7.0 to about 8.0, more preferably about 7.0 to about 7.5, and most preferably is about 7.3.

As in the past at U.S. Patent number 5,135,759 and WO 02/41906 described in, can use one or more to form dye mixture through the DNA of UV or excited by visible light selective dye.The example of the selective dye that excites through UV-light (UV) comprises Hoechst 33342 and Hoechst 33258, and the two all can (St.Louis MO) buys by Sigma-Aldrich.Comprise through the exemplary selective dye of excited by visible light and can purchase Probe, Inc. (Eugene, SYBR-14 OR), and the two benzimide-BODIPY described in the WO02/41906 in Molec μ lar ^Conjugate 6-{[3-((2Z)-2-{[1-(boron difluoride alkyl)-3; 5-dimethyl-1H-pyrroles-2-yl] methylene radical }-2H-pyrroles-5-yl) propionyl] amino }-N-[3-(methyl 3-[({4-[6-(4-methylpiperazine-1-yl)-1H-3 ' H-2,5 '-bisbenzimidazole-2 '-yl] and phenoxy group } ethanoyl) amino] propyl group } amino) propyl group] hexanamide (" BBC ").Use can be used or unite in these dyestuffs each separately; Perhaps, described dyestuff also other can be had the UV of cell permeability and the dyestuff of excited by visible light and unite use separately or with aforementioned dyestuff, as long as can not produce unacceptable detrimentally affect to the viability of spermatid under the concentration that can carry out the described sorting in other places.

Perhaps, also can utilize fluorescent polyamide to form dye mixture, more specifically be to utilize the polymeric amide that is conjugated with fluorescent mark or report.This type of is marked at when being incorporated into nucleic acid can send fluorescence.Be connected with fluorescent mark or report that the polymeric amide example of son comprises, as people such as Best, Proc.Natl.Acad.Sci.USA, 100 (21): 12063-12068 (2003); People such as Gygi, Nucleic Acids Res., 30 (13): 2790-2799 (2002); U.S. Patent number 5,998,140; U.S. Patent number 6,143,901 and U.S. Patent number 6,090,947 in disclosed those, wherein each piece content is all quoted as a reference herein.

Fluorescent nucleotide sequences also can be used for the mark spermatid.This type of nucleotides sequence is listed in and can sends fluorescence when containing the nucleic acid hybridization of target sequence or complementary sequence, but then can not send fluorescence when non-hybridization state.This type of Nucleotide openly sees as U.S. Patent Application Publication No. 2003/0113765 (quoting as a reference) herein.

Sex specific antibodies also can be used for the spermatid in the mark dye mixture.For example, in this embodiment, sex specific antibodies can be puted together in fluorescence part (or reporter molecules of equal value).Because the antigen of antibodies only is present on the cell that has X chromosome or Y chromosome, can optionally identify this type of cell according to its fluorescence (with respect to non-blooming unlabeled cells).In addition, can use respectively more than one the sex specific antibodies that partly links to each other with different fluorescence simultaneously.Thereby can it be distinguished according to the intercellular different fluorescence that have X chromosome or Y chromosome.

Also can use the spermatid in the luminous color selectivity nano crystalline mark dye mixture.These particles are also referred to as quantum dot, as U.S. Patent number 6,322, and 901 and U.S. Patent number 6,576,291 is described is (all quoting as a reference herein) well known in the art.These nanocrystals are puted together in the various biological material, comprise as peptide, antibody, nucleic acid, Streptavidin and polysaccharide (seeing as U.S. Patent number 6,207,392,6,423,551,5,990,479 and 6,326,144, all quote as a reference herein), and be used for the detection of biological target and (see as U.S. Patent number 6,207,392 and 6,247,323, both all quote as a reference herein).

The concentration of DNA selective dye or any other type dye is preferably the function of a series of variablees in the dye mixture, described variable comprise cell for the temperature of the perviousness of selected dyestuff, dye mixture, finish the needed time quantum of dyeing and desired enrichment degree in sorting step subsequently.Generally speaking, preferred dye strength is enough to reach desired dye levels in appropriateness in the short time, and the detrimentally affect that can not produce essence to sperm viability.For example, Hoechst33342, Hoechst 33258, SYBR-14 or the concentration of BBC in dye mixture generally at about 0.1 μ M between about 1.0M, be preferably about 0.1 μ M to about 700 μ M, more preferably about 100 μ M are about 200 μ M extremely.In particularly preferred embodiments, Hoechst 33342, Hoechst33258, SYBR-14 or the BBC concentration in dye mixture generally at about 400 μ M to about 500 μ M, most preferably be about 450 μ M.Therefore, be provided with down at a kind of dyeing condition, the concentration of Hoechst 33342 is preferably about 100 μ M.Being provided with down at another kind of dyeing condition, the concentration of Hoechst 33342 is about 150 μ M.Be provided with down at another kind of dyeing condition again, concentration is preferably about 200 μ M.And be provided with down at another kind of dyeing condition, the concentration of Hoechst 33342 most preferably is about 450 μ M.

Again for example, the concentration of fluorescent polyamide (for example those described in the U. S. application publication number 2001/0002314) is generally about 0.1 μ M to about 1mM, and preferred about 1 μ M is to about 1mM, 5 μ M about 100 μ M extremely more preferably from about, even 10 μ M more preferably from about.

Also can randomly comprise the additive that can strengthen sperm viability in the dye mixture.Exemplary additive comprises as mentioned microbiotic, the somatomedin of having discussed in " collection of cell sample " or regulates in the cell and/or the composition of extracellular oxidation/reduction reaction.Can in collecting liquid, add examples of such additives in view of the above.

In a single day dye mixture forms, and can preserve under the arbitrary temp in the series of temperature scope; Generally speaking, temperature range is about 4 ℃ to about 50 ℃.For example, dye mixture can be stored under the temperature of " low relatively ", promptly temperature is about 4 ℃ to about 30 ℃; In this embodiment, temperature is preferably about 20 ℃ to about 30 ℃, more preferably about 25 ℃ to about 30 ℃, most preferably is about 28 ℃.Perhaps also dye mixture can be stored in " centre " temperature range, promptly temperature is about 30 ℃ to about 39 ℃; In this embodiment, temperature is preferably about 34 ℃ to about 39 ℃, more preferably about 37 ℃.In addition, also dye mixture can be stored in the temperature range of " high relatively ", promptly temperature is about 40 ℃ to about 50 ℃; In this embodiment, temperature is preferably about 40 ℃ to about 45 ℃, more preferably about 40 ℃ to about 43 ℃, most preferably is about 41 ℃.A series of variablees are generally depended in selection for preferred temperature, comprise as cell to the concentration of dyestuff in the perviousness of used dyestuff, the dye mixture, time that cell is retained in dye mixture and in sorting step desired enrichment degree.

Dyestuff in the spermatid picked-up dye mixture can continue sufficiently long for some time, to reach the DNA dyeing of desired degree.The described time generally is enough to make dyestuff to be incorporated into the DNA of spermatid, thereby can both sortings be come according to different and measurable fluorescence intensity between the spermatid that has X and Y chromosome.Generally speaking, this time can not surpass about 160 minutes, preferably is no more than about 90 minutes, also more preferably no more than about 60 minutes, and most preferably is about 5 minutes to about 40 minutes.

Therefore, in one embodiment, it is the dye mixtures of about 100 μ M to the dyestuff of about 200 μ M that formation contains spermatid, mobility inhibitor and concentration, and this dye mixture is kept for some time in about 41 ℃ temperature.In another embodiment, the mobility inhibitor contains 0.204g NaHCO in every 25ml pure water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O0.090mol/L).

In another embodiment, it is the dye mixtures of about 400 μ M to the dyestuff of about 500 μ M that formation contains spermatid, mobility inhibitor and concentration, and this dye mixture is kept for some time in about 41 ℃ temperature.In another embodiment, dye strength is 450 μ M.In another embodiment, the mobility inhibitor contains 0.204g NaHCO in every 25ml pure water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O 0.090mol/L).

Again in another embodiment, it is the dye mixtures of about 100 μ M to the dyestuff of about 200 μ M that formation contains spermatid, mobility inhibitor and concentration, and this dye mixture is kept for some time in about 28 ℃ temperature.In another embodiment, the mobility inhibitor contains 0.204g NaHCO in every 25ml pure water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O0.090mol/L).

And in another embodiment, it is the dye mixtures of about 400 μ M to the dyestuff of about 500 μ M that formation contains spermatid, mobility inhibitor and concentration, and this dye mixture is kept for some time under about 28 ℃ temperature.In another embodiment, dye strength is 450 μ M.In another embodiment, the mobility inhibitor contains 0.204g NaHCO in every 25ml pure water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O 0.090mol/L).

Sorting

The mobility inhibitor also is used in sorting sperms cell stage chien shih cell and does not move.Generally speaking, in case according to the present invention with staining of sperm, can according to any known can according to the isolating method sorting of fluorescence it.Commonly used and known method comprises the flow cytometry system, as U.S. Patent number 5,135, and 759,5,985,216,6,071,689,6,149,867 and 6,263,745; International Patent Publication No. WO 99/33956 and WO01/37655; In U.S. Patent Application Serial 10/812,351 and the corresponding International Patent Publication No. WO 2004/088283 thereof illustrated and describe those, quote its content herein as a reference).When according to these method sortings, sperm is introduced in the mouth of pipe of flow cytometer as sample liquid.Therefore in one embodiment, sample liquid can include dyeing spermatid and mobility inhibitor.

Similar with it, the sheath fluid that is used for parcel sample liquid flow when walking cell instrument also can contain the mobility inhibitor.Generally speaking, utilize forced air or syringe pump sheath fluid to be introduced the mouth of pipe of flow cytometer.Preferred forced air is carbonic acid gas or nitrogen, more preferably nitrogen.Perhaps, forced air also can be carbonic acid gas, but in this case, must be noted that and reduce bubble as far as possible.

Also can randomly contain additive in sample liquid or the sheath fluid, as mentioned microbiotic of discussing in " collection of cell sample ", the adjusting cell is interior and/or the composition or the somatomedin of extracellular oxidation/reduction reaction.Can be in view of the above in these additives each be added in any liquid.

The collection of sorting cells

In case finish sorting, sorting cells be collected in contain in the container of collecting liquid.Generally speaking, the purpose that (employing) collects liquid comprises the influence of buffering collection container to spermatid, or provides the liquid support for cell.

In one embodiment, can contain mobility inhibitor and protein source in the collection liquid.If contain protein source, then it can be any spermatid viability and protein source compatible with used mobility inhibitor of can not disturbing.The example of common protein sources comprises breast (comprising hot homogenate and skimming milk), newborn extract, yolk, yolk extract, soybean protein and soybean protein extract.This type of proteinic working concentration can be about 1% (v/v) to about 30% (v/v), and preferred about 10% (v/v) is to about 20% (v/v), more preferably from about 10% (v/v).

Collect in the liquid and also can randomly contain additive, as mentioned the composition of the interior and/or extracellular oxidation/reduction reaction of microbiotic of discussing in " collection of cell sample ", somatomedin or adjusting cell.Can in view of the above each adding in these additives be collected in the liquid.

Therefore, in a certain embodiment, collect liquid for containing 0.097mol/L NaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The aqueous solution of O and 10% (v/v) yolk, pH is about 6.2, more preferably pH is about 7.0, even more preferably about 6.5.Preferably will collect liquid is kept in the atmosphere that partial pressure of carbon dioxide is higher than air; For example, partial pressure of carbon dioxide can be greater than 0.9 in the atmosphere, and more preferably 0.95, also more preferably 0.99.

Sorting cells can be collected into and contain or be coated with in the container of freezing stretching, extension agent, to replace conventional use of collecting liquid.Therefore, in specific embodiment, sorting cells is collected in the freezing stretching, extension agent that contains the mobility inhibitor.In another embodiment, sorting cells is collected in so freezing stretching, extension agent, and it contains mobility inhibitor, water, Triladyl ^(WI contains glycerine, tris, citric acid, fructose, tylosin 5mg/100ml, gentamicin 25mg/100ml, spectinomycin 30mg/100ml and lincomycin 15mg/100ml for Minitube, Verona), yolk and pyruvate salt.And in another embodiment, collecting liquid is so freezing stretching, extension agent, and it contains 0.097mol/LNaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The aqueous solution of O, and contain 25g Triladyl in every 75ml water ^, 25g yolk and 10mM pyruvate salt.

The freezing stretching, extension of sorting cells

In case finish the sorting of sperm and collect it to collection container, these sperms promptly can be used for female mammal is inseminated.This needs that hardly sperm is carried out other processing and just can carry out immediately.Equally, also can be with sperm cooling or for future use freezing.In this case, add freezing stretching, extension agent, sperm is benefited farthest to reduce cooling or freezing influence to the viability or the back mobility of thawing.

The mobility inhibitor can be used for making the cell in the freezing stretching, extension agent not move.Generally speaking, freezing stretching, extension agent contains mobility inhibitor, protein source and freezing protective material.If contain, for example also can adding, protein source contacts with collection container so that cell support, buffering cell to be provided.Protein source can be any can not disturb the spermatid viability and with the compatible protein source of concrete used mobility inhibitor.The example of common protein sources comprises breast (comprising hot homogenate and skimming milk), newborn extract, yolk, yolk extract, soybean protein and soybean protein extract.This type of proteinic working concentration can be about 10% (v/v) to about 30% (v/v), and preferred about 10% (v/v) is to about 20% (v/v), more preferably from about 20% (v/v).

Preferably contain freezing protective material in the freezing stretching, extension agent, to alleviate or to avoid freezing shock or to keep the fertility of sperm.Numerous freezing protective material known in the art.Be suitable for multiple choices being arranged, depend on and treat that freezing sperm derives from any system with the common freezing protective material that uses of given stretching, extension agent.Suitable freezing protectant example comprise as, glycerine, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, trehalose, Triladyl ^And their combination.If contain freezing protective material, then its amount in freezing stretching, extension agent is generally about 1% (v/v) to about 15% (v/v), and preferably its amount is extremely about 10% (v/v) of about 5% (v/v), and more preferably its amount is about 7% (v/v), and most preferred amount is about 6% (v/v).

In specific embodiments, freezing stretching, extension agent comprises mobility inhibitor, water, Triladyl ^, yolk and pyruvate salt.And in another embodiment, freezing stretching, extension agent is for containing 0.097mol/LNaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The aqueous solution of O, and contain 25g Triladyl in every 75ml water ^, 25g yolk and 10mM pyruvate salt.

In another embodiment, freezing stretching, extension agent comprises mobility inhibitor, water, Triladyl ^And yolk.And in another embodiment, freezing stretching, extension agent is for containing 0.097mol/LNaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The aqueous solution of O, and contain 25g Triladyl in every 75ml water ^, 25g yolk.

Also can randomly contain as mentioned microbiotic, the somatomedin of discussing in " collection of cell sample " in the freezing stretching, extension agent or regulate in the cell and/or the composition of extracellular oxidation/reduction reaction.Can in view of the above each adding in these additives be collected in the liquid.

Above described the present invention in detail, it is evident that, when not breaking away from the scope of the invention of claims definition, can modify and change it.

Embodiment

Provide following non-limiting example further to illustrate the present invention.

Embodiment 1

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and sample is transported to the dyeing facility in 25 ℃ of temp controlled vessels.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, seminal fluid is suspended in TCA cushion or the inhibitor based on carbonate, preparation number pipe contains 150 * 10 ⁶The suspension of sperm/ml.Following Table I has shown compositions for use and dyeing condition.

Table I

The sample name	Moiety	?pH	Concentration (μ M) Hoechst	Temperature (℃)
					10mM?pyr TCA	The TCA that contains the 10mM pyruvate salt	?7.3	?600μM	?28℃
10mM?pyr CO 2	CO 2The TCA that contains 10 mM pyruvate salts of balloon parcel	?7.3	?600μM	?28℃
					Carbonate 6.2	Based on the inhibitor of carbonate, pH6.2	?6.2	?600μM	?28℃
Carbonate 7.3	Based on the inhibitor of carbonate, pH7.3	?7.3	?600μM	?28℃

In sperm suspensions, add the equal portions 10mM Hoechst aqueous solution so that concentration is 600 μ MHoechst.During the dyeing sperm suspensions kept somewhere in 28 ℃ of water-baths (about 1 hour).From dyeing branch sperm suspensions, pipette 50 μ l aliquots, add 25 ℃ of 200 μ l and contain the TCA (pH7.3) of 10mM pyruvate salt with initial static reverse, after at least 5 minutes the starting time, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.The comparative result of tropic movement per-cent is seen Fig. 1-3 before the IVOS.

Embodiment 2

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and sample is transported to the dyeing facility in 25 ℃ of temp controlled vessels.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, seminal fluid is suspended in TCA cushion or the inhibitor based on carbonate, preparation number pipe contains 450 * 10 ⁶The suspension of sperm/ml.Following Table II has shown compositions for use and dyeing condition.

Table II

The sample name	Moiety	?pH	Concentration (μ M) Hoechst	Temperature (℃)
					10mM?pyr TCA	The TCA that contains the 10mM pyruvate salt	?7.3	?1000μM	?28℃
Carbonate 6.2	Based on the inhibitor of carbonate, pH6.2	?6.2	?1000μM	?28℃
					Carbonate 7.3	Based on the inhibitor of carbonate, pH7.3	?7.3	?1000μM	?28℃

In sperm suspensions, add the equal portions 10mM Hoechst aqueous solution so that concentration is 1000 μ MHoechst.Sperm suspensions was kept somewhere in 28 ℃ of water-baths 1 hour, then according to specific description among each figure, was 150 * 10 based on the inhibitor of carbonate with it dilution with the TCA that contains the 10mM pyruvate salt or pH6.2 ⁶The typical sorting concentration of sperm/ml.In scheming the specified time, each from the sperm suspensions of dyeing and dilution, pipettes 50 μ l aliquots, and add 25 ℃ of 200 μ l and contain the TCA (pH7.3) of 10mM pyruvate salt with initial static reverse, after at least 5 minutes the starting time, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.The comparative result of tropic movement per-cent is seen Fig. 4-6 before the IVOS.

Embodiment 3

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and sample is transported to the dyeing facility in 25 ℃ of temp controlled vessels.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, seminal fluid is suspended in TCA cushion or the inhibitor based on carbonate, preparation number pipe contains 450 * 10 ⁶The suspension of sperm/ml.Following Table II has shown compositions for use and dyeing condition.

Table III

The sample name	Moiety	?pH	Concentration (μ M) Hoechst	Temperature (℃)
					10mM?pyr TCA	The TCA that contains the 10mM pyruvate salt	?7.3	?300μM	?41℃
Carbonate 6.2	Based on the inhibitor of carbonate, pH6.2	?6.2	?300μM	?41℃
					Carbonate 7.3	Based on the inhibitor of carbonate, pH7.3	?7.3	?300μM	?41℃

In sperm suspensions, add the equal portions 10mM Hoechst aqueous solution so that concentration is 300 μ MHoechst.Sperm suspensions was kept somewhere in 41 ℃ of water-baths 30 minutes, then according to specific description among each figure, was 150 * 10 based on the inhibitor of carbonate with it dilution with the TCA that contains the 10mM pyruvate salt or pH6.2 ⁶The typical sorting concentration of sperm/ml.In scheming the specified time, each from the sperm suspensions of dyeing and dilution, pipettes 50 μ l aliquots, and add 25 ℃ of 200 μ l and contain the TCA (pH7.3) of 10mM pyruvate salt with initial static reverse, after at least 5 minutes the starting time, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.The comparative result of tropic movement per-cent is seen Fig. 7-9 before the IVOS.

Embodiment 4

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and the carbonate buffer solution of sample with 2 parts diluted, is transported to the dyeing facility in 25 ℃ of temp controlled vessels.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, pipette aliquot carbonate sperm suspensions, with its centrifugal 5 minutes of 500 * g, remove supernatant liquor and precipitation be resuspended in the TCA damping fluid of 41 ℃ of pH7.3, preparation 1mL 150 * 10 ⁶The suspension of sperm/ml.By the aliquot seminal fluid being suspended in 41 ℃ of TCA damping fluids that contain 10mM pyruvate salt, pH7.3, prepare other 1mL 150 * 10 again ⁶Sperm/ml suspension.In sperm suspensions, add the equal portions 10mM Hoechst aqueous solution so that dye strength is 400 μ M Hoechst.During the dyeing sperm suspensions is kept somewhere in 41 ℃ of water-baths.Pipette 50 μ l aliquots from painted sperm suspensions, add the identical cushion of 200 μ l uniform temps, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.IVOS analyzes the results are summarized in Figure 10.

Embodiment 5

Except for the following differences, obtain according to the same procedure of embodiment 4 and prepare sample of sperm: the sperm that is used to suspend is TCA and the TCA that contains 10 μ M vitamin Ks with the cushion that dyes and IVOS analyzes.IVOS analyzes the results are summarized in Figure 11.

Embodiment 6

Except for the following differences, obtain according to the same procedure of embodiment 4 and prepare sample of sperm: the sperm that is used to suspend is TCA and the TCA that contains 100 μ M vitamin Ks with the cushion that dyes and IVOS analyzes.IVOS analyzes the results are summarized in Figure 12.

Embodiment 7

Except for the following differences, obtain according to the same procedure of embodiment 4 and prepare sample of sperm: the sperm that is used to suspend is TCA and the TCA that contains the 1mM Thioctic Acid with the cushion that dyes and IVOS analyzes.IVOS analyzes the results are summarized in Figure 13.

Embodiment 8

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and the carbonate buffer solution of sample with 2 parts diluted, is transported to the dyeing facility in 25 ℃ of temp controlled vessels.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, with sperm suspensions centrifugal 5 minutes of 500 * g, remove supernatant liquor and precipitation be resuspended in the TCA damping fluid of 28 ℃ of pH7.3, preparation 1mL contains 150 * 10 ⁶The suspension of sperm/ml.By the aliquot seminal fluid being suspended in 28 ℃ of TCA damping fluids that contain 10mM pyruvate salt, pH7.3, prepare other 1mL 150 * 10 again ⁶Sperm/ml suspension.In sperm suspensions, add the equal portions 10mM Hoechst aqueous solution so that dye strength is 600 μ MHoechst.During the dyeing sperm suspensions is kept somewhere in 28 ℃ of water-baths.Pipette 50 μ l aliquots from the dyeing sperm suspensions, add the identical cushion of 200 μ l uniform temps, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.IVOS analyzes the results are summarized in Figure 14.

Embodiment 9

Except for the following differences, obtain according to the same procedure of embodiment 8 and prepare sample of sperm: the sperm that is used to suspend is TCA and the TCA that contains 100 μ M vitamin Ks with the cushion that dyes and IVOS analyzes.IVOS analyzes the results are summarized in Figure 15.

Embodiment 10

Except for the following differences, obtain according to the same procedure of embodiment 8 and prepare sample of sperm: the sperm that is used to suspend is TCA and the TCA that contains the 1mM Thioctic Acid with the cushion that dyes and IVOS analyzes.IVOS analyzes the results are summarized in Figure 16.

Embodiment 11

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and the carbonate buffer solution of sample with 2 parts diluted, is transported to the dyeing facility in 25 ℃ of temp controlled vessels.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, pipette aliquot carbonate sperm suspensions, with its centrifugal 5 minutes of 500 * g, remove supernatant liquor and precipitation be resuspended in the TCA damping fluid that 1ml TCA damping fluid or 1ml contain 2.5mM, 10mM, 25mM or 50mM pyruvate salt, preparation 1mL contains 150 * 10 ⁶The suspension of sperm/ml.In sample, add MON33342 solution so that the dyestuff final concentration is 600 μ M.In 28 ℃ of water-baths, hatch suspension.Pipette 50 μ l aliquots from the dyeing sperm suspensions, add the identical cushion of 200 μ l uniform temps, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.The result of tropic movement % as shown in figure 17 before IVOS analyzed.

Embodiment 12

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and the carbonate buffer solution of sample with 2 parts diluted, is transported to the dyeing facility in 25 ℃ of temp controlled vessels.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, pipette aliquot carbonate sperm suspensions, with its centrifugal 5 minutes of 500 * g, remove supernatant liquor and precipitation be resuspended in the 1ml TCA damping fluid, preparation 1mL contains 150 * 10 ⁶The TCA damping fluid of sperm/ml.Pipette a carbonate sperm suspensions, with its in 500 * g centrifugal 5 minutes, remove supernatant liquor and precipitation be resuspended in the TCA damping fluid that 1ml contains the 10mM pyruvate salt, preparation 1mL contains 150 * 10 ⁶The 10mM pyruvate salt TCA damping fluid of sperm/ml.In sample, add the SYBR14 dye solution so that the dyestuff final concentration is 20 μ M.In 28 ℃ of water-baths, hatch suspension.Pipette 50 μ l aliquots from the dyeing sperm suspensions, add the identical cushion of 200 μ l uniform temps, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.The result of tropic movement % as shown in figure 18 before IVOS analyzed.

Embodiment 13

Utilize artificial vagina from the sexual maturity bull, to collect bull semen, and with sample with the dilution of 2 parts carbonate buffer solution, in 25 ℃ of temp controlled vessels, to be transported to the dyeing facility.Seminal fluid is once reception, just according to standard and known method (people Theriogenology such as Farrell, 49 (4): 871-9 (in March, 1998)), utilize Hamilton-Thorn mobility analyser (IVOS) to analyze its concentration, mobility and propulsion.According to seminal concentration, pipette aliquot carbonate sperm suspensions, with its centrifugal 5 minutes of 500 * g, remove supernatant liquor and precipitation be resuspended in the 1ml TCA damping fluid, preparation 1mL contains 150 * 10 ⁶The TCA damping fluid of sperm/ml.Pipette a carbonate sperm suspensions, with its in 500 * g centrifugal 5 minutes, remove supernatant liquor and resuspension and be deposited in 1ml and contain in the TCA damping fluid of 10mM pyruvate salt, preparation 1mL contains 150 * 10 ⁶The 10mM pyruvate salt TCA damping fluid of sperm/ml.In sample, add BBC solution so that the dyestuff final concentration is 100 μ M.In 28 ℃ of water-baths, hatch suspension.Pipette 50 μ l aliquots from the dyeing sperm suspensions, add the identical cushion of 200 μ l uniform temps, (preceding tropic movement %) analyzes by tropic movement per-cent before the IVOS analysis to measure.The result of tropic movement % as shown in figure 19 before IVOS analyzed.

Embodiment 14

Except for the following differences, obtain according to the same procedure of embodiment 4 and prepare sample of sperm: dyeing concentration is 200 μ M BBC.IVOS analyzes the results are summarized in Figure 20.

Claims (33)

1. contain the spermatid suspension of the composition of sperm alive and downward modulation sperm Sugar intake, the sperm concentration in the described suspension is less than about 1 * 10 ⁶Sperm/ml or be at least about 1 * 10 ⁸Sperm/ml.

2. contain the spermatid suspension of the sperm alive that do not move, the sperm concentration in the described suspension is less than about 1 * 10 ⁶Sperm/ml or be at least about 1 * 10 ⁸Sperm/ml.

3. the spermatid suspension that contains sperm alive, this sperm has more the mobility characteristics of sperm in the epididymis with respect to the sperm that penetrates in the same germline body, and the sperm concentration in the described suspension is less than about 1 * 10 ⁶Sperm/ml or be at least about 1 * 10 ⁸Sperm/ml.

4. contain sperm alive, potassium, and randomly contain the spermatid suspension of sodium, the sperm concentration in the described suspension is less than about 1 * 10 ⁶Sperm/ml or be at least about 1 * 10 ⁸Sperm/ml, and the mol ratio of potassium and sodium was greater than each 1: 1.

5. the suspension of claim 4, wherein the mol ratio of potassium and sodium was greater than each 1.25: 1.

6. the suspension of claim 4, wherein the mol ratio of potassium and sodium was greater than each 1.5: 1.

7. the suspension of claim 4, wherein the mol ratio of potassium and sodium was greater than each 1.75: 1.

8. the suspension of claim 4, wherein the mol ratio of potassium and sodium was greater than each 2: 1.

9. contain the composition of sperm alive, downward modulation sperm Sugar intake and the spermatid suspension of DNA selective dye.

10. the spermatid suspension that contains do not move sperm alive and DNA selective dye.

11. contain the spermatid suspension of sperm alive and DNA selective dye, described sperm is with respect to the sperm that penetrates in the same germline body, its metabolic rate and mobility have more the characteristics of sperm in the epididymis.

12. contain the spermatid suspension of the sperm alive that do not move, be combined with the DNA selective dye on the DNA of described sperm.

13. contain the spermatid suspension of sperm alive, for the sperm that penetrates in the same germline body, described sperm metabolic rate and mobility have more the characteristics of sperm in the epididymis, and also are combined with the DNA selective dye on its DNA.

14. the suspension of claim 10, wherein dyestuff is a DNA selectivity fluorescence dye.

15. the suspension of claim 10, wherein dyestuff is that UV excites or the dyestuff of excited by visible light.

16. the suspension of claim 10, wherein dyestuff is from by Hoechst 33342, Hoechst33258, SYBR-14 and two benzimide-BODIPY ^Conjugate 6-{[3-((2Z)-2-{[1-(boron difluoride alkyl)-3; 5-dimethyl-1H-pyrroles-2-yl] methylene radical }-2H-pyrroles-5-yl) propionyl] amino }-N-[3-(methyl { 3-[({4-[6-(4-methylpiperazine-1-yl)-1H; 3 ' H-2,5 '-bisbenzimidazole-2 '-yl] phenoxy group } ethanoyl) amino] propyl group } amino) propyl group] select in the group formed of hexanamide.

17. the sperm suspensions of claim 2 contains carbonate source in the wherein said suspension.

18. the sperm suspensions of claim 2 contains NaHCO in the wherein said suspension ₃, KHCO ₃And C ₆H ₈O ₇H ₂O.

19. the sperm suspensions of claim 2, the self-contained 0.097mol/LNaHCO of wherein said suspension preparation ₃, 0.173mol/LKHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The aqueous buffer solution of O.

20. the sperm suspensions of claim 2, the concentration of sperm is at least 1.25 * 10 in the wherein said suspension ⁸Sperm/ml.

21. the sperm suspensions of claim 2, the concentration of sperm is at least 1.5 * 10 in the wherein said suspension ⁸Sperm/ml.

22. the sperm suspensions of claim 2, the concentration of sperm is at least 1.75 * 10 in the wherein said suspension ⁸Sperm/ml.

23. the sperm suspensions of claim 2, the concentration of sperm is less than about 9.0 * 10 in the wherein said suspension ⁵Sperm/ml.

24. the sperm suspensions of claim 2, the concentration of sperm is less than about 7 * 10 in the wherein said suspension ⁵Sperm/ml.

25. the sperm suspensions of claim 2, the concentration of sperm is less than about 5 * 10 in the wherein said suspension ⁵Sperm/ml.

26. the sperm suspensions of claim 2, the concentration of sperm is less than about 2 * 10 in the described suspension ⁵Sperm/ml.

27. the sperm suspensions of claim 2, the concentration of sperm is less than about 1 * 10 in the wherein said suspension ⁵Sperm/ml.

28. to the painted method of spermatid, described method comprises the dye mixture that formation is such, it contains the potassium of complete spermatid alive, motion inhibition amount, and the DNA selective dye.

29. the method for claim 28, wherein dye mixture is in CO ₂In the atmosphere of dividing potential drop with respect to the air increase.

30. be formed for the method for the spermatid suspension of cell sorting, described method comprises mixes the spermatid source with the composition that suppresses the spermatid mobility, to form spermatid suspension, spermatid concentration is less than about 1 * 10 in the described suspension ⁶Spermatid/ml or be at least 1 * 10 ⁸Spermatid/ml.

31. be formed for the method for the spermatid suspension of flow cytometry method, described method comprises Mammals is penetrated seminal fluid collecting to the damping fluid of the depressomotor that contains the inhibition amount, to form sperm suspensions, contain less than about 1 * 10 in the described suspension ⁶Spermatid/ml or be at least 1 * 10 ⁸Spermatid/ml.

32. the method for claim 30 wherein contains the DNA selective dye in the cell suspending liquid.

33. the suspension of claim 18 also contains the DNA selective dye.

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