GB2399270A - Hysteroscopic insemination system - Google Patents

Abstract

The present invention provides a hysteroscopic insemination system comprising a reservoir element, a catheter system (2) to which said reservoir element is responsive, a plurality of sperm cells contained within said reservoir element and a hysteroscopic compatible media contained within said reservoir element and to which said sperm cells are responsive. The hysteroscopic insemination system of the invention may be used to artificially inseminate such mammalian species as equids, bovides and swine.

Description

a''' I! - 2399270

SYSM OF MACROSCOPIC SEA ON OF AS

This application claims the 'benefit of, and any applicable priority to, U. S. provisional application number 60/238,294, hereby incorporated by reference.

TECHNICAL FIELD

This invention relates generally to the fieldofinsemmation of mammals, Specifically, it relates to systems to achieve insemination which may be particularly applicable once sperm have been treated or processed in some manner, such as sorting the sperm Trough flow cytometry. More particularly, the mver lion may relate to systems to achieve insemination, with a low number of spermatozoa as, compared to conventional artificial insemination, through hysteroscopic insemLnationtechniques. The inventionmay be particularly applicable to systems for ir:-seminating squids, bovids and swine. Furthermore, the inventor may be particularly applicable to sex selection of offspring in mammals.

BACKGROUND

Artificial insemination (AI), in venom fonns, has beenused to achieve successful rates of pregnancy and resulting offspring inmmmals. Traditions forms of AI include various ViYo technologies such as convoutional intrauterine artifci.al iusemmation techniques, ormore particularly, trms cervical artificial insemination. Natural insemination doses may typically be large. For example, in some species, natural insemination may involve sperm numbers on the order of 2-20 x 109 sperm. In contrast, conventional Al techniques in the same species may require sperm numbers once orderof200-SOO x 106 sperm. This con stillbe viewed as a large number of sperm, especially if the sperm are processed some manner. Given Me traditional requirements for high numbers of sperm to achieve acceptable numbers of pregnancies, other inseminationtechniques have been soughtto address developing breeding technologies that may result in a lower sperm count for subsequent insertion. Low dose insemination has been particularly discussed the PCT publication WO 99/33956, entitled "Sex-Specific Insertion of Mammals WiLowNumberof Sperm Cells." Hysteroscopic lusemination has been proposed and to some extent discussed in Moms et al., 20(J0, hereby incorporated by reference.

- ''.]T?':" : i: v I: ! i, . It should be preliminarily noted that all references mentioned in this application, and including additional reference notmeu;tioned, are listed at l}le end of this written discussion, all of which should be considered as incorporated by reference.

High-speed sperm sorting using flow cytometry has been used successfully as a breeding technology to produce offspring in mammals, such as horses, cattle, sheep, rabbits, pigs and hurrahs. It can potentially be used for several other species as well. Technologies have been developed to enhance ormodify the pregnancy and resulting offspring oirnammals, particularly win regard to the processing of spend and insemination techniques.

One potential lmitg factor in optimizing Me success of artificial insemination, as recognized in Pickett et al., l9B9, may be inseminating with low numbers of spermatozoa.

Pickett etal. recognized that apotential minimal recommended dose for conditional artificial inseniination in the mare may be as high as 500 x 106 progressively motile sperm. Win low sort rates of.arod 700 sponnatozoa per second in some spenn sorting technologies, it may take several days to obtainthe recommended dose otspennatozoa for atidcial insernLnation.

This may not only be impractical, but the viability of the spermatozoa may also be sigucandy reduced. Low-dose insemination techniques, therefore, may be a desirability to those skilled irk tle art to potentially reduce the number of spermatozoa needed for acceptable fertility rates.

Additionally, what might be considered low doses for insemination may not typically result in acceptable fertility rates. Fertilizer rates may be considered acceptable or statistically comparable, for example, Whey are achievable over a high sample size, range or percentage of the fertility rates resulting from conventional AI. Lowose insernmation techniques, therefore, may be a deslrabilityto those skilled m the art to potentially reduce the number of spermatozoa needed to maximize fertility.

Whether in Consideration of low-dose msernination or to improve AI fertility rates generally, an additional concern regardlog artificial insemination is the efficiency of the procedure as a whole win regard to the resulting numbers of pregnancies. number of _,:; . .: ''5' À ;- i:: ., procedural steps may have been used in conventional Al procedures, such as We synchror imhon of esters in breeding mares; the preparadon of the insemination dose, snore particularly Me use of extenders for Me dilution (Kenny et al., 1975) and resuspension of spermatozoa(usg TALP or HES-buffered Tyrode's Medium, for example), cenfiging the sample Trough a density or viscosity gradient (using PERCOLL or the like); assessing viability or motility; and the particulars of the insemination procedure. Insemunation procedures have historically included ultrasound and rectally-guided techniques.

Hysteroscopic mseminatior olmares has also been conducted, as described in the Vasquez et al., 1998 and Manning et al., 1998 references. Only [united success, however, has been demonstrated utilizing the above described procedures conventional Al and in the Vasquez et al. and the Meg et al procedures. In particular, neither reference may have produced statistically comparable conception rates to conventional AI. In particular, the Vaso,uez et al. arid Me Manning et al. references may have demonstrated conception rates of 33% for 3.8 x 106 spermatozoa and 22% for 1.0 x 106 spermatozoa, respectively, Inch may be cornered ncomparable conception rates relative to conventional AI for the species involved. Despite the previous and substantial attempts at producing an efficient procedure for AI, heretofore, a long felt but unsatisfied need for an efficient procedure for the hysteroscopic insemination of mammals has existed inbreeding technology. Furthermore, and given Me ditionsl requirements for high numbers of spermatozoa to achieve acceptable numbers of viable pregnancies, as described above, a need for efficient, low-dose hysteroscopic insernmation has heretofore existed in current breeding technology.

Furthermore, deep intra uterine inseniination of swine has been conducted, as desenbed in 1 he Vasquez et aL, 2000 reference. However, insemination was conducted with what might be considered a high number of spermatozoa, on the order of 20 x 107 spermatozoa However, Masques et al., 2000, may have identified the long felt but unsatisfied steed for an insemination procedure to address breeding teclmologies utilizing lower spermatozoa numbers for insemination, such as may result from current spenn sorting technologies.

Therefore, as may have been demonstrated from the Vasquez et al., 1998, Vasquez rLi;.i; 'do :i' '' . ' ' Y.bi: : I..

et al., 2000, and Manning et al., l9Y8, references, those skilled in the art may have failed to address the identified need for providing an rnsemmation tecboique potentially resulting in high fertility rates in mammals and the resulting high production in mammals, high ferocity rates for low insemination dosages, and insertion techniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insemination technique.

One tmportantprocedural step with regard to inseminationprocedure, generally, is the establishment of a insemination dose containing desirable Embers of viable and motile spermatozoa to potentially provide higher fertility rates. Procedures for the selection of motile spermatozoa may have been conducted with regard to conventional Al, for example in the reference Grondahl et al., 1996 and ire hysteroscopic msemnabon generally, by establishing adensity orviscosity gradientutilg, forexample, PERCOLL (SigmaChemical Co., St. Louis, RIO) alone or in conbuation with other substances. However, heretofore the substantial efforts to fractionate viable or motile sped eve not particularly addressed the identified needs for an insemination technique potentially providing high fertility rates, high I 5 fertility rates for low insemination dosages, and unseminationtechniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insemination technique.

A second poteral issue with regard to insemination procedure, generally, is the establishment of a insemination dose containing desirable numbers of viable and motile spermatozoa to potentially provide higher fertility rates without a particular motility test, as described above. The introduction of a density or viscosity gradient may introduce a stress to the spermatozoa that may actually reduce the actual number of viable and motile spermatozoa available Dom a particular sample. Heretofore, the substantial efforts to Fractionate viable or motile spenn have not particularly addressed the identified needs for an insemination technique providing for high fertility rates, high fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insemination technique. In fact, due to the potential for reduction n the total renumber of viable or motile spenn Tom a particular inseminate sample, previous attempts incorporating a density or viscosity gradient mall have actually taught away from the present procedure of providing for an insemination technique ? - d;: which may require less efforts to obtain a viable, low-dose insemination sample A third potential issue win regard to insemination procedure, generally, is the establishment of a compatible volume for Me particular insemination technique. One recognized need, as described above, is the desire to potentially provide higherfertility rates.

A second recognized need, also described above, is the ability to use low numbers of spermatozoato potentially achieve hightertilityrates. The insemination dose volume may be determined by the particular insemination technique. However, the dose volume may contain a desirable number of spermatozoa to potentially provide for a higher rate of fertility.

Substantial attempts have been made to establish an appropriate insemination technique that would allow for the appropriate number of spermatozoa, given the potential volume requirements of We insermTatioD technique, to potentially provide acceptable fertility rates.

However, determ ning a compatible insemination dose volume for a particular iusem nation technique, to fiercer potentially provide higher rates of fertility, has potentially not been established for hysteroscopiG mseminAtior, as theroported fertility rates in Vasquez et al. and ManDg et al. may demonstrate.

Additionally, and as previously mentioned, the sperm sample may be processcdprior to the insemination procedure. Conventional AI, for example, may provide for the use of extenders for the dilution (Kenny et al., 1975) and resuspension of spermatozoa. However, the particular media used may not be compatible with the insermnabon procedure itself.

Incompatibility of Ho sample media may result in lower deposition numbers of spermatozoa or dose volume or a lower fertility rate. Furthermore, Me mode or form of the deposited inseminationdose or the particularmethod of deposit during Al may further affectthenumber of deposited spermatozoa available for conception. However, heretofore the substantial efforts directed toward insemination media may have not particularly addressed He need for providing an insemination technique potentially resulting in high fertility rates, high fertility rates for low insemination dosages,, and insemination techniques to address issues of efficaciousness, particularly up regard to the hysteroscopic insemination technique.

An additional factor to consider is He timing of insenur ation. Insemination timing, s i / } W;. ..: may be an important factor, for example, to sperm viability and longevity and the timirtg of the estrous cycle ofthe mammal. Particularly at issue might be the distant location of sperm sample acquisition (i.e the location of the male marnrnal) and ultimate location of the AI.

Previous efforts may have been made in conventional AI to preserve the sperm sample prior to insemination and to coordinate the insemination win Me estrous cycle. However, heretofore the substantial efforts directed to mseation timing may have not particularly addressed the identified needs for, and may have even failed to understand the problems of, prowding an insemination technique potentially resulting in high fertility rates, high fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly win regard to the hysteroscopic insemination technique.

The source of the sperm sample may also be of importance to the resulting insemination. Epididymal acquisition ofthe sperm sample (obtaining sperm sample ire epididyms ofthe testis; ductules emerging posteriorly Dom the testis that holds sperm during maturation and that forms a tangled mass before uniting into a single coiled duct which is continuous with the vas deferens) may provide some inherent advantages as to tim ng ofthe insemination and viability ofthe speml. However, heretofore the substantial efforts directed to sperm source, viability and mserrunation timing may have not fully addressed the identif fed needs for, and may have even failed to understand Me problems of, providing au insemination technique potentially resulting in high fertility rates, high fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to dle hysteroscopic irsemnation technique.

DISCLOSURE OF TE INVENTION

The present inventors have recognized the potential problems associated with conventional methods of artificial insemination. Accordingly, embodiments of the present invention may provide for the production of a mammal through Me use of artificial insnationthatmay address inadequacies of previous insertunationtechniques and systems.

7 he invention may comprise, according to particular embodiments, a method of producing a mammal whereby potentially high fertility rates may be accomplished, fertilibr rates which may be statistically compatible win conventional AI results, and potentially high fertility rates win We use of low spermatozoa doses. More particularly, embodiments of the present invention may provide for the production of a renal through the use of hysteroscopic nsmunation techniques. Additionally, the present invention may comprLse embodiments particularly directed at mammals such as equids, bovids, and swine, among other mammals. Embodiments ofthe present invention, therefore, may even be considered development away from previous efforts of artificial insemination.

One object of the present invention-, therefore, is to provide for the production of a rrn utilizing an efficacious, procedure. Therefore, a goal of the present invention is to provide a technique of artificial 7emintion for mammal production suchthatlowerrumbers of spermatozoa may be used in We insemination dose relative to conventional AI and other insemination techniques, while, in particular embodiments of We invention, at least statistically comparable success rates in fertility are maintained.

Another object of We present invention is to provide for the production of a mammal utilizing an artificial irmernination procedure that may potentially achieve high fertility rates consistent with lower spermatozoa production Dom breeding teetnologies such as sperm sorting. A goal of We present invention, therefore, is to provide a technique of artificial insemination for mammal production that achieves statistically comparable success rates in fertility, compared to conventional AI arid other insemination techniques, with lower-doses of spermatozoa.

Furthermore, an object of We mventon is to provide for We production of a mammal utilizing an artificial insemloation procedure that may enhance steps involved in the artificial insemination. To this end, one goal of the present invention is to provide a technique of artificial insemination for maternal production such that steps of estrous cycle timing, spermatozoa source, viability, longevity and processing, insenation dose media and volume, and insemination timing may be optimized, particularly for low-dose insemination and potentially high fertilizer.

An additional objectofepresentvention, according toprefe:Ted embodiments, is toprovidefortheproductionofamammalthroughhysteroscopic insemination A goal ofthe present invention, therefore, is to provide a technique of artificial insemination for mammal production such Mat the insertion of the insemination dose, guiding ofthe insenunation dose to the deposition site, deposition of the insemination dose at the appropriate location, in an appropriate mode or form, may be accomplished to achieve other objects and goals as previously stated. In accordance vath particular embodiments of the invention, blister insemination and bubble or Both insemination may be introduced as preferred cmbodunents to optimize fertility rates. A particular goal of the present invention is to provide a technique of artificial insemination for mammal production utilizing a catheter comprising a videoendoscope for guiding and depositing We insemination dose.

Additionally, an object of the present invention, ire accordance with particular embodiments, isto provide for the production ofvanous mammal species utilizing an artificial insemination procedure. A goal of Me present invention, therefore, is to provide a technique of artificial insnination for mats such as equids, bovids and swine, among over species.

A fisher goal is to provide a technique of artificial insemination for various mammal species that additionally provides for low numbers of spermatozoa in the insemination dose and for potentially high fertility rates, particularly rates that may be statistically comparable to conventional AI.

Over obj cots of He inversion are disclosed rouout other areas ofthe specification and claims. II} addidon, Be goals and objectives may apply either in dependent or independent fashion to a variety of other goals and objectives in a variety of embodiments.

BRIEF DESCRIPTION OF 111E DRAWINGS

Figure 1 is a partially exploded and perspective diagrammatic view of the optical element, candela, catheter and syringe in accordance with one embodiment of the present invention.

Figure 2 is a diagrammatic depiction of the reproductive organs of a female of a rramTnalian species and, in particular, a depiction of artificial insemination in accordance with one embodiment of the present invention.

Figure 3 is amagnifed diagrammatic view of an enshrouded insemination insertion embodiment of the present invention.

MODE(S) FOR CARRYING OUT DIE PRESENT INVENTION The basic concepts of the invention may be embodied in many different ways. The inventive concept may involve the materiads, elements, apparatus, device and methods for We production of a mammal Dough artificial inse unction. Furthermore, while one preferred embodiment ofthe invention may be particularly directed to tile production of elands through artificial insemination, the broad concept of Me Invention should be construed as a disclosure of the production of mammals general, and as indicated, to other mu 1 species such as bovids and swine.

As should be uIllerstood, the present invention includes a varied of aspects Mat may be used in various combinations depending upon the application's needs. Ibe invention is intended to encompass a variety of embodiments of mammal production and combinations thereof. It involves bow methods and devices to accomplish the various aspects explained.

In addition, while some methods and devices are disclosed, it should be understood that these may be varied. Importantly, as to all of the foregoing, all aspects should be understood to be encompassed by this patent both independently and in combination as set form in the claims now or later issued.

AccordLngly, one embodiment ofthe present invention may provide for the collection of sperm cells from a male of the species of interest In accordance win one embodi.nent, sperm cells are collected Mom one or more stallions ofthe equine species. According to this embodiment, semer1 may be collected, and in preferred embodiments, semen may be collected with a commercially available artificial vagina, perhaps Mom at least one stallion of known acceptable fertility. An artificial vagina such as one made available by Animal Reproduction Systems may be used with an n-line gel filter, and inpreferred embodiments, used on alternate days throughout collection. After collection, the semen may be evaluated for gel-free volume, motility, arid spenn concentration. In accordance with another preferred embodiment, sperm cells may be collected from other male species of mammal, particularly that of bovids, equids or swine. An alternative embodiment of the present invention may provide for the collecting of epididymal sperm cells obtained from the epididymis of the testis of He male species of the mammal. The alternative embodiment proYidtog for the use of epididymal sperm Ray be incorporated with all over disclosed embodiments herein, either in single or in combination.

Furthermore, the present invention provides particular embodiments a hysteroscopic insemination sample comprising a reservoir element, a catheter system to which the reservoir element is responsive, and a plurality of epididymal sperm cells contained withinthe reservoir element.

After sperm collection, an artificial insemination sample may be established for the insemination of the female species. In accordance with one embodiment of the present invention, the sample may be prepared as, having a low number of sperm compared to a natural insemination dosage for the mammal. The sample may have a low number of spenn for particular breeding technologies, and in accordance with preferred embodiments, the sample may have a low number of sperm as the result of sortLng He sperm for particular sexed sperm. In accordance with a preferred embodiment of the invention, He spermatozoa may be stained win Hoechst 33342 and sorted into X and Y chromosome-beanng populations based on DNA content using a commercially available SX MoFlo spend sorter.

Additionally, an artificial insemination sample may be established at volumes, in accordance with prefened embodiments, at volumes between about 30 and 150 ul, less Ulan about 500 al, about 230 al, and about 100 ul. One embodiment of He present invention is directed to establishing a hysteroscopic inseminatiorl compatible volume, preferably an insemination sample at a volume selected Dom a group consisting of: between about 30 and ul, less than about 500 ul, about 230 ul, and about 100 ul. Purermore, the present invention is directed to a hysteroscopic insemination sample comprising a reservoir element a catheter system to which Me reservoir element is responsive, and a hysteroscopic compatible volume of sperm contained within said reservoir element. The hys$eroscopic compatible volume of spend contained within said reservoir element may compuse a volume selected from a group consisting of: between about 30 and lSO ul, less Man about 500 ul, about 230 ul, and about 100 ul.

Next, in accordance with preferred embodiments of the invention, the artificial insemination sample may be placed within a catheter or catheter system. In accordance with alternative embodiments, the sample may be placed within a reservoir element or other sample holdLng element responsive to the catheter or catheter system. A catheter or catheter system should tee understood to define any device, system ormethod of irsertionto canals, vessels, passageways, or body cavities to perIrut j action or withdrawal of fluids, and in accordance with preferred embodiments such injection or withdraw may provide the response of lChe reservoir element or sample holding element, or to keep a passage opera Preferably, the catheter or catheter system may be used in conjunction with a guide element, and in preferred embodiments an optical element or device, and in preferred embodiments an illumination element, to provide guidance In the artificial msemunation procedure, as more particularly described below. Preferred embodiments may provide the Probing of the illumination element. However, manual guidance may also be implemented.

In accordance with preferred embodiments of the invention, and as depicted in Figure 1, the msemmation sample may be aspirated into an equine GIFT catheter (2) (Cook Veterinary Products, Brisbane, Australia) using preferably a 6-mL disposable syringe (4) attached to the jectionport (6) on the distal end (8) ofthecaeter. The loadedcathetermaybe withdrawn into a tube, or preferably an outer polypropylene cam}ula (lO), Which may be responsive to an optical element (12), and in accordance with preferred embodiments, passed down a working channel of a Pentax EPM 3000 videoendoscope (Pentax UK Ltd. Slough, Bucks UK), Accordingly, an instance, time or duration may be determined in which the artificial insemination may be appropriate. In accordance with preferred embodiments of the present TO 028311 P6TSOI/023U4 invention, a detenunabon may be made as to an esbous time for a female mnunal. In accordance with an embodiment of the invention, estrus may be induced to determine the estrous time and, for multiple mares, even synchronized. Esbus may be defined as a state which Me female mammal is capable of conceiving and estrous cycle may be defined as the correlated phenomena of the endocrine and generative systems of a fete mammal, potentially from the being of one period of estrus to not later than We beginning of the next. In accordance with a preferred embodiment; estrus may be induced, and for multiple female mammals synchronized, by alministeringa substance dachas aprogestagen, preferably for mares albenogest, and preferably 10ml orally, potentially for 10 consecutive days, followed by 250 tug cloprostenol i.m., potentially on day 11. Furthermore, a female mammal may be induced into ovulation at the time of insemination. Ovulation may be induced, and in preferred embodiments, by the administration of 3000 in human Chorionic Gonadotropin (hCG, Chorulon, Intervet, Inc., Millsboro, Collared), preferably aninisteredinhavenously at the time of insemination or up to approximately 8 hours previously. Ovulation may even be induced, in preferred embodunents, by the admnlsbationof from about 2000 to about 5000 iu human Chononic Gonadotropin.

In preferred embodiments, estrous cycles may be synchronized by adndnistermg a synthetic progestagen altrenogest (0.044mglkg p.o., Regumate; Hoeclast Roussel Vet, Warren, New Jersey, USA) daily for 10 consecutive days. Luteolysis may be induced, in preferred embodiments with the prostaglandin analogize, cloprostenol (250,ug Esumate, i.m.; Bayer Corporation, Agriculture Division, Shawnee Mission, Kansas, USA) ministcTcd on the eleverth day.

A time when the female mammal is appropriately Refile may be detemuned. The ovaries may be exarruned, and in preferred embodiments examined ultrasonoaphical}y, and preferably every second day until a follicle, and in accordance with preferred embodiments a dominant follicle, preferably of> 30mm diameter, is detected. The female may be exaIruned until a follicle, preferably pre-ovulatory, of preferably 35mm is detected. In one embodiment, the female may be inseminated during the same day as estrous inducement or synchronization, the same day as ovulation inducement or synchroration, or Me same day as estrus and insemination inducement or sychronization.

Next, In accordance with particular embodiments ofthc invention, the guide element, and in preferred embodiments the optical element (12), may be vaginally inserted into the female. Furthermore, the catheter (2), and in preferred embodiments the reservoir element, may be lnseTted into the female. The sequence of insertion ofthe optical element (12) and Mecatheter (12) may be sequential or coincidental in time.

The guide element or optical element (12) and the catheter (2) may then be guided through the vagina (20) of tile female, as depicted in Figure 2. In accordance win one embodiment, the optical element (12) and catheter (2) may be manually guided. In fact, the catheter (2) may be manually guided without the optical clement (12). However, accuracy in finding the UTJ and the potential result. increased fertility rates, particularly for low numbers of sperm and overall potential efficacy in the procedure, may require a more accurate guidance procedure. Inpreferred embodirnerlts, the optical element or endosoope, preferably a flexible endoscope, in preferred embodimentshaving dunensions of 1.6 m long wianouter diameter of 12 mm, may be guided through We cervix (22) and propelled forward Tough Me uterine lumen (24), or in additional embodiment", through a uterine horn of the female of the species. An added benefit of Me use of Me videoendoscope can be lack of a need to rectally guide the insemination process, as may have been required in past efforts by those of slill in the art.

The uterotubal junction (I) (30) of the female D]Ga1 may Men be located, preferably optically with the optical element (12). The catheter (2) may then be positioned m the vicinity of or proximate to the uterotubal junction. In accordance with preferred embodiments, the endoscope may be directedunder visual control along the uterine horn (26) ipsilateral to the ovary containing the pre-ovulatory follicle (28). Preferably the tip of the 2S endoscope may be directedproximate to, and in preferred embodiments within about 3-5cm of said uterotubal junction, and in preferred embodiments within about 3 -5 cm of the papilla (32) of the uterotubal junction.

At least a portion of the artificial insemination sample may then be exuded from Me catheter (2). In accordance with preferred embodiments, the outer cannula (10), followed by the inner GIFT catheter (V containing the spemt suspersion, may be exQuded from We working channel of the endoscope until the tip of the GIFT catheter touches the uteroWbad inaction, and in preferred embodiments, touches the papilla. In accordance with alternative embodiments of the present invention, a portion or at least a portion of the artificial insemination sample may be aspirated during extrusion from We catheter, thereby potentially creating al1 aspirated sample. Such aspirated samples should be understood to include bubbled samples and Browed samples. An aspirated sample may not only provide better adherence to a surface in We vicinity of the UTJ, but may further allow for improved fertility rates.

Furthermore, an embodiment of the present invention provides a hysteroscopic insemination element comprising a uterus of a female species of a mammal, a plurality of sperm cells contained within said uterus, and an aspirated volume of media surrounding or interspersed win the sperm cells and to which the sperm cells are responsive Such alternative 1 5 embodiments should be construed as disclosed with regard to all embodiments of Represent invention, either in single or in combination, and should be construed to be disclosed as such.

A low number of sperm may be deposited in the vicinity of lbe uterotubal junction.

In accordance with preferred embodiments, a plunger ofthe syringe (4) may be depressed to deposit the sample, and in particular embodiments, a small volume (preferably perhaps 1001) of We sample, preferably onto We surface ofthe papilla The guide element, and in preferecl embodiments the optical element (12), and the catheter (2) may be withdrawn Bom Me uterus of Me female mammal. IA accordancowithpreferred embodiments, filtered air may be introduced within Me uterus of the female to facilitate passage of Me instruments through the uterine lumen. Furthermore, in accordance with preferred embodiments, the filtered air may be evacuated from the uterus, preferably sunultaneous to Me withdrawal ofthe optical element (12) and catheter (2).

The placing of a low number of spenn may be accomplished, in one preferred embodiment of the invention, by placing with the catheter (2) a number of spenn, preferably numbers selected from: less then about ten million sperm, fess then about five million sperm, less than about two million sperm, less than about one million sperm, less than about five hundred thousand sperm, and less fh about one hundred thousand sperm.

Subsequently, the fertilization of an egg of the female may occur and the production of en offspring mammal from the fertilized egg. In accordance with preferred embodiments of the invention, success levels of fertilization may be statistically comparable to a convendoDal uterine body artificial insemination process. Statistically comparable success I levels may be defined as fertilization rates of at least about 75% success rates, at least about 65% success rates, at least about 60% success rates, at least about 50% success rates, at least about 45% success rates, and at least about 90% of a success rate practically experienced with conventional Al for any particular species. Furthermore, success levels for the present invention may be statistically comparable to a conventional uterine body artificial insemination process over a sample of cumulative fertilizations which is greater then about 100, which is greater then about 500, and which is greater than about 1000. Success rates may further statistically provide for at least a confidence level of at least about 95 percent confidence (potentially expressed as P> or equalto aboutO.05), therefore potentially teeing statistically comparable with conventional AI.

Additionally, success rates for We present invention may potentially have Me same P value over a range of differing spennnumbers, potentially such as 1, 5 or to x 106 sperm for equine.

Furthermore, a success level or rate may be expressed in terms of sample size, whereby the present invention may provide, in preferred embodiments, any ofthe aforementioned success rates over a power calculation (,B) of at least about 80 percent. Additionally, the success rates, in preferred embodiments, given particular insenunation doses for sex-sorted stallion spermatozoa, may even apply to a low number of spermatozoa and may routinely produce fertility rates of at least about 90% of hose rates resulting from conventional artificial insemination for a species. Additionally, according to particular embodiments of the present invention, the previous success rates may even be achieved for species of mammal such as bovids, squids, and swine.

In regard to spoon vabili ty, longevity and mobility, for example, the present invention may provide for establishing an artificial insemination sample utilizing flesh sperm. The tenn i "fresh sperm" may be broadly defied as spend that teas not been treated, processed or preserved in alar manner such that the sperm viability, longevity andJor mobility might be compromised. Such treatment, processing or preserving may include, for example, the sorting of sperm, the freezing arid subsequent thawing of sperm, the dilution and resuspension of sperm, and motility and viability testing or separation, generally, and in particular, Percoll gradient processing. The use of fresh sperm ire accordance with embodiments of the present invention herein may permit the use of low numbers of sperm for,ention even, for example, when using pOOr quality collected semen.

Altemative embodiments of the present invention may broadly provide for treatment, processing and preserving of Insemination sperm. AIternative embodiments of the present invention may provide, for example, for the selection of the collected sperm cells more likely to achieve insemitiom Accordingto one embodimentofthe present invendonthe selection of desired sperm cells or sperm cells more likely to achieve insemination may compose concentrating the more motile sperm collected. Additional embodiments may provide the step of centrifi:gg the spend through density gradients, and in preferred embodiments a Percoll gradient A potentially prefelTed embodiment may use a gO:4s% Percoll gradient. An additional embodiment may comprise limiting the concentration to less than about twice the starting concentration. Implementation of the Percoll gradient should be understood to encompass We useofPercollpreorpastprocessingofthe sperm, and inparticular, potentially pre orpost sorting. Therefore, irk accordance wad embodiments herein, Me Percoll gradient procedure may be used with "flesh spend." The sample established from the desired or selected sperm cells may be used to establish the artificial msernination sample, potentially increasing the fertility rates due, at least in part, to the potentially higher rate of viability of Me sample. The Percoll gradient procedure in accordance with the present invention may be conducted inconjunctionwiththe use of lower numbers of spermplacedwithinthe depositing catheter, as more particularly described supra.

Additionally, establig al. artificial isemmatiou sample may be provided by establishing a sample having hysteroscopic compatible media, thereby providing for potentially increased fertility rates and an efficacious insemination procedure. More particularly, an embodiment ofthe inventionmay provide establishing an artificial insemination sample, and in preferred embodiments having a low number sperm compared to natural inseniirmtion doses, and providing for the establishment of an artificial insemination sample compatible media' for example extender, and in preferred embodiments, a skim milk medium, such as EZ Mixin CST) (Animal Reproduction Systems, Chino, CA), preferably as a diluting media, potentially prior to additional processing. In accordance with preferred embodiments ofthe presentinvendon, dilution may occur to no more then a2:1 ratio, to no more then a 5:1 ratio, and to no more than a 10:1 ratio, to potentially achieve at least a hysteroscopic compatible volume ormediawith appropriate concentrations. Furthermore, embodiments ofthe present invention may provide for the use of an extender, potentially a second extender provided after spenn processing and potential ly in conJunctionwth a first extender, as previously mentioned, to establish the sample utilizing hysteroscopic compatible media. The second extender may serve to resuspcud the sperm sample after processing, and more particularly, provide for a sample utilizing hysteroscopic compatible media. According to preferred embodiments, Me medium or second extender may comprise a TALP medium, a HEPES-buffered Tyrode's medium, and an Androhep medium. In accordance withpreferred embodiments ofthe present invention, dilution may be performed with a slam milk medium such as EZMixin CST@, with a TALP medium, with aHEPES-buffered Tyrode's medium, and with an Androhep medium, either single or in combination, to potentially achieve at least a hysteToscopic compatible volume or media with appropriate concentrations.

An additional embodiment of the present invention may also provide for aI1 artificial insemination sample utilizing ah,rsteroscopic compatible mediaormediumhavng diameter coordinated viscosity. The viscosity may, for example, potentially facilitate the steps of exuding and depositing the sample. Furthermore, in accordance with potentially preferred embodiments of the present invention, the use of compatible media or medium may create an artificial insemination sample having a viscosity of preferably greater than about that ofthe blood of said mammal or greater than about that of a saline solution. Furthermore additional embodiments may provide for compatible media having a viscosity of greater than about 1 OOcp, a media having a viscosity of greater Man about 3 OOcp, a media having a viscosity of greater than about 1 OODcp, a media having a viscosity of greater than about 3000cp, and a media having a viscosity of greater than about 6000cp, each at about Me mammal's average body temperature.

Furermore1 an embodiment of the present invention may provide a hysteroscopic inserruration sample comprising a reservoir element, a catheter system to which said reservoir element is responsive, aplurality of sperm cells contained within said reservoir element; and hysteroscopic compatible media contained within said reservoir element and to which said sperm cells are responsive. In accordance with additional embodiments, an insemination containment wherein said plurality of sperm cells are contained within said reservoir element may Include a low number of spenn cells as compared to the number of spenn cells typically found in a natural insemination.

Altering a property of the insemination specimen or spend cell sample may be conducted according to Me present invention, and in accordance with preferred embodiments, determining an estrous time for a female of a species of said mammal and Bert altering a property of said insemination specimen to establish an artificial insemination sample at about said estrous time. Alternative embodiments mayprovide altering a properly ofthe sample at about the time determined for the female of said species to be appropriately fertile, as previously defined. The present invention may also provide initiation of the altering of a property of the insemination specimen within a time selected from: within about twenty- four hours of said time determined for said female of said species of said mammal to be appropriately fertile, wiinabouttwelve hours of said time determined for said female of said species of said mammal to be appropriately fertile, within about eight hours of said time detested for said female of said species of said mammal to be appropriately fertile, within about Free hours of said time determined for said female of said species otsatd mammal to be appropriately fertile, and within about one hour of said time determined for said female of said species of said mammal to be appropriately fertile. This alteration may consist of prepanug the sample, sorting the sperm. thawing the spend, or Me like.

One particular embodiment of Me present invention may provide for establishing a hysteroscopic compatible media and the concentration of sperm to select sperm more likely to achieve insemination. Particularly, one potentially preferred embodimertofe invention may provide preparation of the semen through centrifilgabon. The semen may be diluted to provider for example, 100 x 106 spermatozoalrnl in preferably a commercial slain milk extender (EZ-dn CST@, AD0nsl Reproduction Systems, Chino, CA). The sperm suspension may be protected from light and maintained for preferably 6 hours at room (20 25 c) temperature to simulate the potential time needed to sort the spermatozoa, if so desired.

The sperm suspension may then be centrifuged through a 90:45% Percol1 (Sigma Chemical Co., St. Louis, MO, USA) discontinuous density gradient with the goal of reconcetrabngthe cells end to potentially select ahithly motile fraction of spermatozoa. The 90 K Percoll may be diluted at a ratio of 1:1 (ply) with HEPES-buffered Tyrode's medium (Grendahl e' al., I 0 1996) to make a 45% solution. In a preferably 1 5-ml, centrifuge tube, preferably 1 mL of I 45% Percoll may be carefilIy layered on top of preferably I my of 90% Percoll. Preferably 1 mL ofthe spenn suspension (100 x l o6 spennJrnL inE;-Mixu, CST)may belayered ontop Of Me Percoll layers, and We tube may be centrifuged at 800 x g for a preferred period of 12 minutes. After centrifigation, the supematant may be completely removed "d Me pellet may be resuspended in preferably 6001 EI13PES-buffered Tyrode's Medium. The sperm concentration may be determined, in accordance with one embodiment, using a Densimeter (534B MOD-1, Animal Reproduction Systems, Chino, CA) and Me potential volume to deliver 5 million spermatozoa Potentially of a preferred 1001l1) may be calculated and prepared for insemination In accordance with a broad aspect of the present invention, one embodiment thereof may provide for the positioning ofthe catheter near the UT] ofthe female species of mammal whereby Me catheter is inserted under a surface in the vicinity of the uterotubal junction. At least a portion of Me sample may be exQuded or deposited in the vicirity of Me llTJ under the surface.

One embodiment ofthe present invention may provide for the collecting of sperm cells Lom the male species of mammal, establishing an artificial insemination sample utilizing at least some of the sperm cells collected, and placing the artificial insemination sample LO a catheter. A determination of a time when the female is appropriately fertile may be determined, as described above. The optical element ( 12) and the catheter (2) may be inserted vaginally and guided through the vagina, as depicted in Figure 2. The UTJ may be optically located win optical element (12).

The catheter may be luserted under a surface (34) in the vicinity of the UTJ (30), as depicted in Figure 3. At least a portion of said artificial insemination sample may be extruded under the surface within the vicinity of the UTJ, thus creating a "blister" with Me sample enshrouded between layers. Deposition of at least a portion of the artificial insemination sample may be provided under the surface (34) in the vicinity of the UTJ. The surface (34) may comprise the endometrium or other portion of the uterus or the uterine living. In accordance vthonepreferredembodiment, thecathetermaybe insertedsuchataparticular portion of the uterus is not pierced, and in accordance with one embodiment, such that a vascularized portion of the uterus is not pierced. A vascularized portion of the uterus that may not be pierced, according to one particular embodiment, may include, for example, We mesometrium or the myometrium portion of the uterus or other vascularized portions.

Thereafter, the optical element and catheter may be withdrawn from the female and fertilization of an egg of the female may occur after which production of an offspring mamm al may be expected Tom Me fertilized egg.

An embodiment ofthepresent invention may also provide apotential1y corresponding insemination catheter having a guide element, or in preferred embodiments a videoendoscope or acacuula, areservoir element responsive to the guide element, an extrusion element, or in preferred embodiments a syringe to which said reservoir element is responsive, and a cellular piercing tip (36) positioned Dont of the reservoir element. The catheter may further provide a pierce depth control element, such as an adjustment element or a stop on the piercing tip (36) positioned the vicinity ofthe tip. One embodiment of an insemination catheter of the present invention is depicted in Figure 3.

As previously described and in accordance win the insemination system of ex rusion and deposition under a surface in the vicinity of the UTJ described above, a number of steps of producing a mammal may be performed. A low number of sperm may be placed Me catheter (2), and in preferred embodiments, preferably numbers selected from: less than about ten million sperm, less than about five million sperm, less than aboutwo million sperm, less than about one million sperm, less dean about five hundred thousand spend, and less than about one hundred thousand sperm. Furthermore, the fertilization of an egg may be performed in accordance with the preferred embodiments of Me invention wherein success levels success levels offertilizationmay be statistically comparable to a conventional uterine body artificial insemination process. Statistically comparable success levels may be defined as previously mentioned.

Furennore, the sperm cells may be collected from a male species of mammal, in alternative embodiments ofthe invention, comprising bovids, equids, or swine. In accordance with alternative embodiments of the invention, sperm cells may be selected from collected cells for those cells Mat may be more likely to achieve insemination, as previously described.

Accordingly, an irsemination containment element may be provided, in accordance with embodiments of the invention, preferably comprising a cellular base surface, and in particular embodiments a uterine tin ng OT, in accordance m preferred embodiment, a nonvascularized portion ofthe uterus, such as the mesometrium or Me myomenium, a cellular cover surface adjacent to the cellular base surface, and in particular embodiments, the endometrium or uterine 1 ning, a substantial enshrouded volume between the cellular base and the cover surface, and in preferred embodiments located in the society ofthe UTJ, and sperm cells fiom the male of the species. Preferred embodiments may also utilize low numbers of sperm relative to natural,emination, located within the volume and a sperm emission element adjacent the volume through which sperm may pass. The sperm may be collected, selected, of an inseminate volume, perhaps even of an epididymis origin, or of any other lirnitabon previously discussed. The sperm ermssion element may comprise breach in the endometrium surface of the uterus, as depicted in Figure 3, or may simply occur by diffusion or the like.

Additionally, other potentially independent procedures may be incorporated into Me present invemior and may still be considered as origin the scope of Me present inventiorl.

Such procedures may include sorting the sperm cells by a sex characteristic, thereby establishing a sex-sorted artificial insertion sample, and Inpreferred embodiments having a low number of Earn compared to a natural msemmation dosage for said mammal, may include establishing a low dose sex-selected artificial insemination sample. Furthermore, preserving or freezing, and the subsequent Hawing of, sperm sells may be accomplished in particular embodiments, particularly in regard to various mammals such as equid, boYid and swine. Deposition ofthe insemination sample, may Reprocessed oraspirated In any way, may be deposited with the crypts or folds of He l)TJ, and may provide some type of preservation of the sperm for subsequent insemination. Establishment of an inserrnnation specimen or insemination sample at ahysteroscopic compatible volume and uiling compatible mediamay further provide for allowing cooling of the specimen or sample at room temperature.

Centrifugation may preferably beperformedtb;rough aPercoll gradient for aboutfiveminutes at about 200g and for about ten minutes at about 800g. In accordance with a preferred embodiment, concentrating We more motile sperm may be limited to concentrating to less than about twice the staring concentration. In particular, We broad and narrow concepts embodied in the present invention should be construed as applying to other species of mammal, including equids, bovids and swine. Finally the present invention, directed in part to the producing of an offspring mammal, may farther be considered to disclose an embodiment of an animal produced utilizing a process as described in any of the foregoing method claims Sorting, accordance with embodiments of the present invention, may particularly provide for collect ing sperm cells from a male of a species of mammal, sorting the sperm cells according to a sex-pecific characteristic, establishing a sorted, sex-specific artificial insemination sample, placing the sorted, sex-specific artificial inseabon sample in a catheter; among the various other aspects of the invention disclosed herem that might be incorporated in method of producing a marmnal.

As can be easily understood hom the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. It involves both inse mnahontechniques as well as apparatus to accomplish appropriate iuseniination. In this application, the insemination techniques are disclosed as part of the results shown to be achieved by the venous devices described and as steps which are inherent to utilization. They are simply the natural result of utilizing the devices as intended and described In addition, while some devices are disclosed, it should be Understood that these not only accomplish certain methods but also can be varied in a number of ways huportandy, as to all of the foregoing, all of these facets should be

understood to be encompassed by this disclosure.

The discussion included in this application is intended to serve as a basic description.

The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not Filly explain the generic nature ofthe invention and may not explicitly show how each feature or element can actually be representative of a broader function or of a great variety of alternative ox equivalent elements. Again, these are implicitly Lncluded in this disclosure. Where the slvention is described in device-oriented terminology, each clement ofthe device implicitly performs a fimction. Apparatus claims may not only be included for the devices described, but also method or process clams may be included to address the fimctions the Invention and each element performs. Neither the description nor the terminology is intended to limit Me scope of the clanns which will be included in a filll patent application.

It should also be understood that a variety of changes may be made without departing Tom the essence ofthe invention. Such changes are also implicitly included in the description.

They still fall within the scope of this invention.

Further, each of&e various elements ofthe invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood that as the disclosure relates to elements of the invention, the words for each element may 1Oe expressed by eiivalent apparatus teens or method terms -- even if only the function or result is the same. Such equivalent, broader, or even more generic terms should be considered to be encompassed in the description of each element or action. Such terms can be substituted where desired to make explicit the implicitly broad coverage to which this Invention is entitled As but one example, it should be understood that all actions may be expressed as a means for taking that action or as an element which causes that action. SirnilarIy, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. Regarding this lot aspect, as but one example, the disclosure of an "extruded' should be understood to encompass disclosure of the act of "extruding" -- whether explicitly discussed or not - and, conversely, were there ordy disclosure of Me act of "extruding", such a disclosure should be understood to encompass disclosure of an "extrudes" and even a "means for extruding". Such changes and alternative terms are to be understood to be explicitly included in the description.

Any acts of law, statutes, regulations, or rules mentionedinthis applicationforpatent, or patents, publications, or other references mentioned in this application for patent, are hereby incorporated by reference. In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood as incorporated for each term and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictionary, second edition are hereby incorporated by reference. However, as to each of Me above, to the extent that such information or statements incorporated by reference nught be considered inconsistentwithtle patenting ofthis/these invention(s), such statements are expressly not to be considered as made by Me applicant(s).

Thus, the applicant(s) should be understood to have support to claim at least: i) each ofthe insemination devices asherendisclosed and described, n) the relatedmethods disclosed and described, iii) similar, equivalent, and even implicitvariations of each of these devices and methods, iv)those alternative designs which accomplish each of the fimctions shown as are disclosed and described, v) Hose alternative designs and methods which accomplish each of the Unctions shown as are implicit to accomplish that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, and ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanylug examples, and x) the various combinations and permutations of each of the elements disclosed.

Further, if or when used, the use of the transitional phrase "comprising" is used to rnamtain the "open-end" claims herein, according to traditional claim interpretation. Thus, unless, the context requires otherwise, it should be understood that the term "comprise" or variations such as "composes" or "composing", are intendedto Imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. Such terms should be interpreted in Weir most expansive form so as to afford the applicant the broadest coverage legally permissible.

It should also be notedthat the tenn';at feast one" as used in the following description and claLms is not intended nor used in this disclosure to mean that other claims or descriptions not incorporating the "at least one" language cannot further include one or more like elements. More specifically, the language "at least one" is not intended nor used to change "open-ended" claims, irreverently including devices or methodshaving additional elements or steps apart from those claimed, into "closed- ended"claunswhereindevices ormethorlshavmg additional elements would not be covered by such claims. Accordingly, if or when used, the use of the transitional phrase "comprising" is used to maintain the "open-end" claims herein, according to traditional claun interpretation.

I. PATENT DOCUMENTS _.

DOCUMENT NO. DATE /COUNTRY CLASS SUBCLASS ElLING DATE 5,13 5,759 08104t92 Johnson 424 561 04/26191 _ _. _ _ _. . 6,071,689 _ 06J06/00 Seidel et e1. 4;5 2 OIJ29198 601238,294 05110/00 Morns et al. 05110100 WO 98134094 0608198 NZ. _ II. OTTER DOCUMENTS (Indudlag Author, Title, Date, Pertinent Pages, Etc.) Brasher, V. and ADen, W.R, 'rVideoendor.copic Examination of the Mare's Utau5: Findings in Normal Fertile Mares", Equine Vetelinary JOUTTIai, Vol. 24 (1992), pp. 274-278 Buchanan, B.R, at al, "Insemination of Mares with Low Nunibers of Either Unsexed or Sexed Spermatozoa", Tneriogenology, Vol. 53, pp 13331344, (2000) Caslicl, B.A., "Ihe Vulva and the Vulvo-vaginal Orifice and its Relation to Genital Health of the Thoroughbred Mare", Cornell Veterinarian, Vol. 27, 1937, pp. 178-t87 C ran, D.G., et al, Production of Lambs by Low Dose Intrauterine Insermnatioo with Flow Cytonetrically Sorted and Unsorted Semen", Theriogenology, Vol. 47, pp. 267, (Abstract), (I 997) Fugger, E.F., "Clinical Experierlce with Flow Cytometric Separation of lIumarr X- and YChromosome Beonug Sperm", Tberiocoology, Vol. 52, pp. 1435-1440 (1999)

_

Grondshl, C., ct al, 'In Vitro Production of Equine Embryos' Biology of Reproduction, Monograph Series [, pp. 299-307 (1995) Johnson, IDA. and Welch, G.R., USex Preselection; High- speed flow cytometric sorting of X and Y sperm for maximum efficiency", Ihenogenology, Vo}. 52, (1999), pp. 1323-1341 Johnson, L..A., "Advances in Gender Preselection in Swine" Joumal of Reproduction and Fertility Supplement, Vol. 52,pp. 255-266 (1997) Johnson, L..A., 'Sex Preselection in Swine: Altered Sex Ratios in Of Pspring Following Surgical Insemmadon of Flow Sorted XsndY-BeariDgSperrn", Reproductior.in Domestic Animals,Vol. 26,pp. 309-314 (1991) Johnson, L A., et al., "Sex Preselection in Rabbits: Live Birds from X and Y Spend Separated by DNA and Cell Sorting" Biology of Reproduction, VoL 41, pp. 199-203 (1989) Manning S.T., et al., "Dcvdoprnent of Hysteroscopic semmation of the Uterine Tube in the Mare", Proceedings of the Annual Meeting of the Society for Thenogenology, 1998, pp. X4-B5.

Morris, L.H., et al., "Hysteroscopic insemination of Ail numbers of sperrnatowa at the uterotubai junction of preovulatoryrnares-, Journal afReproduction and Fertility, VaL 118, pp. 95-100 (2000) Parrish, J.J., et al., 'Capacitation of bovine sperm by hepann' Biology of Reproduction, Vol. 38, pp. 1171-1180 (198B) Peippo, J., et al., "Sex diagnosis of equine Reimplantation embryos using the polymerize ehein reaction", lleriogerology, Vol. 44 619-627 (1995) 3 0 Pickett GW, et al., 'Management of the mare for rnexirnum reproductive efficiency" Bulletin No. 6 Colorado State University, Ft. Collins CO. (1989) Schenk, J.L., "Cryopreserv&tionofflow- sortedbovinespermatowa", Iheriogenology, Vol.52,1375-1391(1999) _ _.

Schmid ILL., at al, "Fertilization with Sexed Equine Spermatozoa Using lntracytoplacrnic Spend Injection and Oviductal Irseminatiorl I, 7th International Symposium On Equine Reproduction, pp. 139 (Abstract) (1998) _.

3 5 Scidd, (3.E. Jr, et al., "Artificial Inserron&tion of Heifers win Cooled, Unfrozen Sexed Semen ", Iheriogenology, Vol. 49 pp. 365 (Abstract) (1998) Seidel, G.B. Jr, at al., 'insemination of Heifers with Sexed Spcnn ", Thoriogenology, Vol. 52, pp. 14071421 (1999)

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Squires, RL, 'lBarlylLnbryonicLoss"inBquineDiagnosffcUltrasonography, ladled pp 157163 BdsRantrnen& Mcnnon. Willis and Wig a, Bdffmorc, Maryland (1Y98) Squires, B.L.., et al, "Cooled and frozen stallion sernenn, Bulletin No. 9, Colorado State University, Ft. Collins, CO. (1999)

_

VDzquez, J., at al., "A.I. in Swine; New Sby for Deep Insemination with Low Number of Spennatozoa Using a Non-surgical Methadologyn, 14. International Congress on Animal Reproduction, Vol. 2, Stockhlorn, July, 2000, p. 28 .

_

Vazguez, J., et al., "Nonsurgicial Utcro-tubal Insemination us the Mare", Proceedings Annual MeedDg of the Society I O Vazquez, 1., et al., "Successful Low-Dose Insemination by a Fiberoptic Bndoscopc Technique in the Sow ' Proceedings Annual Conference of the Intemational Embryo Transfer Society, Netherlands, lberiogenology, Yol. 53, January, 2000, pp 201.

Vazquez, J., at aL, "Development of a Non-surgical Deep Intra Uterine Lnsemination Technique", IV International Conference on Boar Semen Preservation, Maryland, August, 19. 9, p 35 sad photo of display board.

Vazquez, J., et al., "Development of a Non-surgical Deep Intra Uterine Insemination Technique", Boar Semen Preservation IV, IVth Internedionrd Conference on Boar Semen Preservation, Maryland, pp. 262263,

_ _

Vazquez, J., et al., 'ypoosmotic Swelling Test as Predictor of the Membrane Integrity in Boar Spermatozoa", Boar Semen Preservation IV IVth International Conference on Boar Sunen Preservation, Maryland, pp. 263.

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Jobuson, L., et al, "Sex Preselection m Swine: Flow Cytornctric Sorting of X- and Y- Chromosome searing Sperm to Produce Offspring", Boar Sentry Preservation IV, 200O, pp. 107-S 14.

Rath 1:) et al., "Low Dose Ioseminadon Technique in the Pig", Boar Semen Preservation IV, 2000, pp. 115 - I 18.

Lindsey, A., et al., '1Iysteroscopic Insemmation of Mares with Nontrozen Low-dose UnsexecS or Sex-sorted Spermatozoa", curentlyutipubli.shecS, pp. 1-15.

Claims (1)

1. Claims for Divisional 3 In the following claims, the numbering system from

   the parent case has been left intact.

   Additions to the claims (if any) are represented by an underline. Deletions Mom the clauns (if any) are represented by a I. 164. A hysteroswpic insemination sYstem comprising: a a reservoir element; , b. a catheter system to which said reservoir element is responsive, c. a plurality of sperm cells contained within said reservoir element; and d. hysterscopic compatible media contained dustbin said reservoir element and to which said sperm cells are responsive.

   165. An msenination containmeahysteroscopic insemination system as described in claim 164 wherein said plurality of sperm cells contained wil bin said reservoir element comprise a lovr nuTnber of sperm cells as compared to a the number of sperm cells typically found in a natural insemination.

   166. An insemination contnmentdeysteroscopic insemination system as described in claim 165 wherein said low ncrnber of sperm cells as compared to a the number of sperm cells typically found in a natural insemination comprises a number of spenn cells selected from a 3roup consisting of: less than about ten nonillion sperm, less than about hire million sperm, less than about two million sperm, less then about one million spew, less {ban about five hundred thousand sperm, and less than about one hundred thousand spend.

   167. An insemination containment e}eEaerhYsteroscopic insemination system as described in claim 166 wherein said sperm cells comprise sperm cells from a male of a species of a mammal selected Mom a group consisting of; bovids, equids, or swine.

   168. An inseminatii elemenvsterosconic insemination system as described in claim 166 wherein said plurality of sperm cells confined within said res,ervor element comprise sec-selected sperm cells Mom a male of a species of mammal. 2t

   -

   169. An nGeirBati YSbroSCOPiC isseminaban system as described in claim 168 wherein said sex-selected sperm cells firm a male of a species of mammal comprise sperm cells firm a male of a species of a mammal selected Dom a group consisting of: bovids, squids, or swine.

   170. A iesnat con";nment elemes,rsteosconic insemination system as described u' claim 164 wherein said hysterscopic compatible media contained within salt esewoir element and to which said sperm cells see responsive comprises a skim mills medium.

   171. A nsasmntion costar elemcnthrsmroscopic insemination system as described in claim 164 wherein said hysterscopic compatible media contained within said reservoir element and to which said sperm cells are responsive comprises a median selected fiom a Coup consisdug of a TALP medium, a ;PES-buffered Tyrode's medium, and an asdrohp medium.

   172. A insemination contain inept elenthYsteroscopic insenunation system as described in claim 164 wherein said hysterscopic compatible media contained within said reservoir element and to which sand sperm cells are responsive comprises a catheter coordinated viscosity media.

   173. A insertion containment clemcnthysteroscopic insemination system as desenbed in claim 172 vLerem said catheter coordinated viscosity media comprises a media which creates an artificial insemination sample having a Viscosity of greater than about that of the blood of said mammal.

   174. A - Cremation containment elementhYsteroscopic insertion system as described in claim 173 wherein said catheter coordinated viscosity media Bother composes a media which creates an artificial insemination sample having a viscosity of greater than about that of a saline solution.

   175. A Hen containment clemcnthysteroscopic insemination system as described in claim 172 wherein said catheter coordinated viscosity media filer comprises a media selected from a group consisting of:: a media having a Viscosity of greater than about lOOcp, a media having a viscosity of greater than about 300cp, a media having a viscosity of greater than about lOOOcp, a media bang a viscosity of greater Man about 3000cp, and a media having a viscosity of greater than about 6000cp, each at about the mammal's average body temperature. * * *

   183. A hysteroscopic insemination sample comprising.

   a a reservoir element; b. a catheter system to which said reservoir element is responsive; and c. a hysteroscopic compatible volume of sperm contained withm said reservoir element.

   184. An insemination containment element as described in claiTn 183 wherein said hysteroscopic compatible volune of sperm contained within said reservoir element comprise a low number of sperms cells as compared to a the number of sperm cells typically found in a natures insenunation.

   185. An insemination continent element as described in claim 184 wherein said low number of Sperm cells us compared to a the number of sperm cells Wpically found in a natural insemination comprises a number oisperm cells selected from a group consisting of: less than about ten million sperm, less than about five million sperm, less than about two nonillion sperm, less than about one million Berm, less than about five hundred thousand Sperry, and less than about one hundred thousand sperm.

   186. An insemination containment element as described in claim 185 wherein sand sperm cells comprise sperm cells from a male of a species of a mammal selected Bom a group consisting of: bovids, equids, or swine.

   187. A hysteroscopic insemination sample as desc;nbed in claim 183 wherein, said hysteroscopic compatible volume of sperm contained within said reservoir element comprises a volume selected from a group consisting of: between about and 150 ul, less than about 500 ul, about 230 ul, and about 100 ul.

   188. An insemuation containment element as described in claim 183, 187 wherein said hysteroscopic compatible volume of sperm contained vathi;a said reservoir element comprise sex-selected spmm cells from a male of a species of mammal.

   189. An insenunation containment element as described in claun 188 wherein said sex- selected sperm cells from a male of a species of mammal comprise sperm cells Mom a male of a species of a mammal selected from a group consisting of: bovids, equids, or sine. * * *

   215. A hysteroscopic insemination sample comprising a reservoir element; b. a catheter system to which said reservoir demerit is responsive; and c. a plurality of epididymal sperm cells contained within said reservoir element.

   216. An insernmation containment element as described in claim 215 wherein said plurality of epididymal sperm cells contained within said reservoir element comprise a low number of sperm cells as compared to a the number of sperm cells Epically found in a natural insertion, 217, An insemination containment element as described in claim 216 vherem said low number of sperm cells as compared to a the number of sperm cells typically found in a natural insemination comprises a number of sperm cells selected Mom a group consisting of: less In about ten million sum, less than about five million spurn, less than about two million sperm, less than about one million sperm, less than about five hundred thousand spume, and less than about one lmndred thousand sperm. 3t

   218. An insemination containment element as desenbed in claim 217 wherein said sperm cells comprise sperm cells from a male of a species of a mammal selected Mom a group consishog of: bovids, equids, or swine.

   219. An insemination continent element as described in Giant' 217 wherein said plurality of epididymal sperm cells contamedwthin said resenroireleme comprise sex-selected sperm cells hom a male of a species of mammal.

   220. An insemion containment element as described ir' clam 219 wherein said sex- selected sperm cell" Bom a male of a species of compose sperm cells from a male of a species of a mammal selected Mom a group consisting of: bovids, squids, or swine. $ * *

   296. An hyseeroscopic insemination Panoply produced in accordance avid the method of any of the foregoing claims. * * *

   309. A method of producing a mammal substantially as hereinbefore described with reference to the accompanying drawings.

   310. An insemination containment element substantially as hereinbefore described Vita reference to the accompanying drawings.

   311. An lusemination catheter substantially as einbefore described with reference to the accompanying drawings.

   312. An insemination sample substantially as herembefore described with reference to the acconpanyg drawings.

   313. A hysterosoopic unserninadon sample substantially as herembefore described with reference to the accornpaDying drawings.

   314. me produced by a method substantially as hereinbefore described with reference to Me accompanying drawings.

   315. An animal produced using a process ubstalldally as hereinbefore described with reference to the accompanying drawings.