GB2399292A - An insemination catheter - Google Patents

Abstract

The present invention provides an insemination catheter (2) for use in producing a mammal through artificial insemination. The catheter (2) comprises a guide element (12), a reservoir element responsive to said guide element (12), an extrusion element to which said reservoir element is responsive and a cellular piercing tip (36) positioned in front of said reservoir element. The catheter of the invention may be used in the insemination of such mammation species as equids, bovides and swine.

Description

SYSTEM OF HYSTEROSCOPIC SEA ON OF AS

This application cla ur s the benefit of, and any applicable priority to, U.S. provisional application number oO/238,294, hereby incorporated by reference.

TECHNICAL FIELD

This invendonrelates generally to We fieldofinsemmationoimammals Specifically, itrelates to systems to achieve insemmationwlichmay beparcularly applicable once sperm have been treated or processed in some manner, such as sorting the sperm through flow cytoney. More particularly, the invention may relate to systems to achieve insemination, with a low number of spermatozoa as compared to conventional Biracial insemination, through hysteroscopic inseninationtechniques. The inventionmay be particularly applicabl to systems POT inseminating equip, bovids and swine. errnore, the invention may be particularly applicable to sex selection of offspring in mammals.

BACKGROUND

Uncial iasehon(AI), invanousfo,basbeenusedtoachieve successfialras Of pregnancy and resulting offspnug in mammals. Traditional forms of AI include various in vitro technologies such as conventional intrauterine artificial insemination techniques, ormore particularly, bans 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 spend. In contrast, conventional Al techniques in the same species may require sperm numbers on the order of 200-500 x 106 sperm. This can still be viewed as a large number of sperm, especially if the sperm are processed in some manner. Given the traditional requirements for high numbers of sperm to achieve acceptable numbers of pregnancies, other insemination techniques have been sought to address developing breeding technologies that may result in a lower sperm count for subsequent insemination Low dose insemination has been particularly discussed in the PCT publication WO 99/33956, entitled "Sex-SpecificInseminationofMammals WithLowNumberofSperm Cells." Hysteroscopic insemination has been proposed and to some extent discussed in Moms et al., 2000, hereby incorporated by reference. ,,?q

It shooed be preliminarily noted Mat all references mentioned this application, and including additional reference not mentioned, are listed at the end of this written discussion, all of which should be considered as incorporated by reference.

Hith-speed sperm sorting using flow cytometry has been used suwessffilly as a breeding technology to produce offspring in mammals, such as horses, cattle, sheep, rabbits, pigs and humans:. It can potentially be use for several over species as well. Technologies have been developed to enhance ormoditythepregnancy end resulting offpungofrnammals, particularly with regard to the processing of sperm and insolation techniques.

One potential ltmg -factorin optimiz ng the success of artificial insemination, as record in Pickett et al., 1989, may be inseminating with low numbers of spermatozoa.

Pickettetal. recognizedtihat apotential minimal recommended close for conventional ardúical insemination in the mare may be as high ad 500 x 106 progressively motile sperm. With low sort mtes of amused D Spermatozoa per second -in some sperm sorting technologies, it may take several days to obtain Me recommended dose of spermatozoa for artificial insemination.

Ibis may not only be impractical, but the viability ofthe spermatozoamay also be significantly reduced Low-dose inserninatioD techniques, therefore, may be a desirability to those skilled lathe art to potentially reduce thenumberofspermatozoaneeded for acceptable fertility rates.

Additionally, what might be considen:d low doses for insemination may not tropically result in acceptable fertility rates. Fertility rates may be considered acceptable or statistically comparable, for example, if they are achievable over a high sample size, range or percentage of the fertility rates resultulg Dom conventional AI. Low-cost insernmtion techniques, therefore, may be a desirability to those skilled in the art to potentially reduce the number of À spermatozoa needed to maximize ferdli.

Whether consideration of low-dose insemination or to improve AI fertility rates generally, an additional concern regarding artificial unsemmation is the efficiency of the procedure as a whole win regard to the resulting numbers of pregnancies. A number of it; . .; Ares procedural steps may have been used in conventional AI procedures, such as the synchrorntion of estrus in breeding mares; the preparation of the insenation dose, more particularly He use of extenders for the dilution (Kenny et al., 1975) and resuspension of spermatozoa(usmg TALP or HEPliS-buered Tyrode's Medium, for example), cenifilng the sample through a density or viscosity gradient (using PERCOLL or the like); assessing viability or moonlit; and the particulars of the insenunation procedure. Insemnabon procedures have historically included ultrasound and rectally-guided techniques.

Hysteroscopic insemination of mares has also been conducted, as described in the Vasquez et al., 1998 and IvIanning ct al., 19g8 references. Only limited success, however, has been demonstrated utilizing the above described procedures in corventional Al and in the Vasquez et al. and He Manning et al. procedures. In particular, neither reference mar have; produced sadistically comparable conception rates to conventional AI. In particular;, He Vasquez et al. and the Manning et aL references may have demonstrated conception rates of. I 33% for 3.8 x 106 spermatozoa end 22% for 1.0 x 106 spermatozoa, respectively, which may I be cowered nconparable eonceplon 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 hysteroscopicinsenunationofmamnalshas existedinbreedingtechnology. Furthermore, and given the traditional requirements for high numbers of spermatozoa to achieve acceptable numbers of viable pregnancies, as described above, a need for efficient, low-dose hysteroscopic insen.unation has heretofore existed in current breeding technology.

Furthermore, deep intra uterine insemination of swine has been conducted, as described in the Vasquez et al., 2000 reference. However, inseminationwas conducted win what might be considered a high number of spermatozoa, on the order of 20 x 10' spermatozoa However, Vasquez et al., 2000, may have identified the long felt but unsatisfied need for an insernmation procedure to address breeding technologies utilizing lower spermatozoa Embers for insemination, such as may result from current sperm sorting technologies.

Therefore, as may have been demonstrated from the Vasquez et al., 1998, Vasquez v.

et.7 20, and Maurg et al., 1998, references, those skills in the may have faded to address the identified need for providing an insemination technique potentially resulting in high fertility rates in mammals and the resulting high production in mammals, him fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insemination technique.

Oneimportantprocedmalstepwithregardto inseminationprocedure, generally, is the establishment of a insemination dose containing desirable numbers of viable and motile spermatozoa to potentially provide higher fertility rates. Procedures for We selection of motile spermatozoa may have been conducted with regard to conventional AI, for example in the reference Grandahl et al., lg96 and in hysteroscopic irsemmation generally, by establi King a density or viscosity gradient utilizing, for example, PERCOLL (Sigma Chemical Co., St. Louis, hlO) alone or in combination with other substances. However, heretofore We substantial efforts to fiactivabe viable or motile sperm have not pardeularly addes,sed tile identified needs for an mseTrunation technique potentially providing high fertility rates, high fertility rates for low insemination dosages, and inserninationtechniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insemination technique.

A second potential 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 We spermatozoa that may actually reduce the actual number of viable and motile spermatozoa available from a particular sample. Heretofore, the substantial efforts to fractionate viable or motile sperm have not particularly addressed the identified needs Or an irsesrunation 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 in the total number of viable or motile sperm from a particular inseminate sample, previous attempts incorporating a density or viscosity gradient may have actually taught away from the present procedure of providing for an insemination technique Or - : which may require less efforts to obtain a viable, low-cost insemination sample third potential issue with regard to insemination procedure, generally, is the establishment of a compatible volume for the particular insemmadon technique. One recognized need, as described above, is the desire to potentially provide higher fertility rates.

second recognized need, also described above, is the ability to use low numbers of spermatozoato potentially achievehiBhtertilityrates. The insemination close 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 Me insem nation technique, to potentially provide acceptable fertility rates.

However, determining a compatible insemination dose volume for aparticularinsennation technique, to fiercer potentially provide higher rates of fertility, has potentially not been established for hysteroscopic lDsen=ation, as the reported fertility rates in Vasquez et al. and Manning et al. may demonstrate.

Additionally, and as previously mentioned, the sperm sample may be processed prior to the insermnation procedure. ConventioDa1 Al, for example, may prwide for the use of extenders for the dilution (Kenny et al., lg7S) and resuspension of spermatozoa. However, the particular media used may not be compatible with the insemination procedure itself: Incompatibility of the sample media may result in lower deposition numbers of spermatozoa or dose volume or a lower fertility rate. Furthermore, the mode or form of the deposited inseminationdose orthe particularmethodofdepositdunug Al mayfirtheraffectthenumber of deposited spermatozoa available for conception. However, heretofore the substantial efforts directed toward insemination media may have not particularly addressed the 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 with regard to the hysteroscopic insemination technique.

An additional factor to consider is the tong of lusernination. Insemination timing s : ,: C'. ! . may be an important factor, for example, to spenn viability and longevity and the timing of the estrous cycle of the mammal. Particularly at issue might be the distant location of spenn sample acquasition (i.e. the location of the male mammal) 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 with the estrous cycle. flowerer, heretofore the substantial efforts directed to insemination timing may have not particularly addressed the identified needs for, and may have even failed to understand the problems of, providing an insemination technique potentially resulting in high fertilizer rates, high fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to Me hysteroscopic insemination technique.

Lee source of the sperm sample may also be of importance to We resulting insemination. Epididymal acquisition ofthe sperm sample (obtmug sperm sample fromthe epididymis ofthe testis; ductules emerging posteriorly from the testis that holds sperm dming 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 timid g of the insemination and viability ofthe sperm. However, heretofore We substantial efforts directed to spend source, viability and insemination tong may have not fillly addressed the identified needs for, and may have even failed to understand the problems of, providing an insemination technique potentially resulting in high fertility rates, high fertility rates for low insemination dosages, and insemination techniques to address issues-ofefficaciousness, particularly with regard to We hysteroscopic insemination technique.

DISCLOSINGS OF THE 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 He use of artificial insemination that may address inadequacies of previous insemination techniques and systems.

I-he invention may comprise, according to particular embodiments, a method of producing a mammal whereby potentially highlertility rates may be accomplished, fertility rates whichrnay be statistically compatible with conventional AI results, and potentially high fertility rates win the use of low spermatozoa doses. More particularly, embodiments of the present invention may provide for the production of a mammal through the use of hysteroscopic insemmtion techniques. Additionally, the present invention may comprise embodiments particularly directed at mammals such as equids, beside,, and swine, among other mammals. Embodiments ofthepresentinvention,therefore, may even tee considered development away fromprevious efforts of artificial insemination.

S)ne object of the present invention, therefore, is to provide for the production of a mammal utilizing an efficacious procedure. Therefore, a goal of the present invention is to provide a technique of artificial insemination [M production suchthatlowernhers of spermatozoa IDay be used in the insemination dose relative to conventional AI and other Lnseminationtechniques,while, inparticularembodiments oftheinvention, atleast statistically comparable success rates in fertility are maintained.

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

Furthermore, an obj ect of We invention is to provide for the production of a mammal utilizing an artificial insemination 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 mammal production such that steps of esbous cycle tinting, spennatozoasource, viability, longevity and processing, inseminationdosemedisandvolume, and insemination tinting may be optimized, particularly for low-dose insemination and potentially high fertilitr.

An additional object ofthepresertinvention, according to preferred embodiments, is toprovidefortheproductionofamammalthroughLysteroscopic insemination. A goal ofthe present invention, therefore, is to provide a technique of artificial insemination for mammal production such that the insertion ofthe insemination dose, guiding ofthe insemination 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 With particular entodimenls of the invention, blister insemination and bubble or froth insemination may be introduced as preferred embodiments to optimize fertility rates. A particular goal of the present invention is to provide a technique of artificial insertion for mammal production utilizing a catheter comprising a mdeoendoscope for guiding and depositing the insem nation dose.

Additionally, an object of the present invention, in accordance with particular embodiments, isto provide for the productionofvariousmammal species utilizing en artificial insemination procedure. A goal of the present invention, therefore, is to provide a technique of artificial msnation for mammals such as equids, bovids and swine, among over species.

Afirther goal is to provide atechnique of artiDcialinsenunationforvariousrnammal species that additionally provides for low numbers of spermatozoa the insemination dose and for potentially high fertility rates, particularly rates that may be statistically comparable to conventional AI.

Oerob}ects of einventionare disclosedthrouthoutotherareas ofthe specification and clauns. addition, the goalsandobjecvesrnayapply Liberia dependentorindependent fashion to a variety of other goals and objectives in a variety of embodiments.

B10EP DESCRIPTION OF TElE DRAWINGS

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

Figure 2 is a diagrammatic depiction of the reproductive organs of a female of a mammalian species and, in particular, a depiction of artificial inserninion in accordance win orre embodiment of the present invention Figure 3 is amagT if fed diagrammatic view of an enshrouded insemination insertion embodiment of the present invention.

MODES) FOR CLOG OR POSER ION The basic concepts of the invention may be embodied in many different ways. The Lnventive conceptmay involvethe materials, elements, apparatus, device andmeods for the production of a mammal through artificasl insemination. Furthermore, while one preferred embodiment of the invention may be p articularly directed to the production of equids through artificial insemination, the broad concept ofthe inventiorlshould be construed as a disclosure of the production olmammals in general, and as indicated, to other mammal species such as bovids and swine.

As should be understood, the present invention includes a variety of aspects that may I 5 be used in venous comblna$ions depending upon the application's needs. The invention is intended to encompass a variety of embodiments of marginal production arid corabinations thereof It Devolves both 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 ofthe foregoing, all aspects should tee understood to be encompassed by this patent both independently and in combination as set forth in the clanns now or later issued.

Accordingly, one embodunert of the present invention may provide for the cot faction of sperm cells Dom a male of the species of interest. In accordance with one embodiment, spew cells are collected from one or more stallions of the equine species. According to this embodiment, semen may be collected, and inprefeTred embodiments, semenrnaybe collected with a commercially available artificial vagina, perhaps Dom at least one stallion of known acceptable fertility. An artificial vagina such as one made available by Animal Reproduction Systems may be used win an in-line gel filter, and inpreferred embodiments, used on alternate daysrououtcollection. Aftercollection, the semenmay be evaluated for gelfree volume, motility, and sperm concentration. In accordance with another preferred embodiment, sperm cells may be collected from other male species of mammal, particularly that of bovlds, squids or swine. alternative embodiment of the present invention may pronde for the collecting of epididymal spermcells obtained *tom the epididymis ofthe testis of Me male species ofthe mammal. The alternative embodiment providing for the use of epididymal sperm may be incorporated with all other disclosed embodiments herein, either in single or in combination.

Furthermore, We present invention provides particular embodiments a hysteroscopic msemmation 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 insemmabon sample may be established for the insemination of the female species. In accordance with one embodiment of the present mvendon, 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 sperm for particular breeding technologies, and in accordance with preferred embodiments, the sample may have a low number of sperm as the result of sorts the sperm for particular sexed sperm. In accordance untie a preferred embodiment of the invention, Me spermatozoa may be stained with Hoechst 33342 and sorted Into and Y cbromosome-beanng populations based on DNA content using a commercially available SX MoFlo spenn sorter.

Additionally, an artificial insemination sample may be established at volumes, in accordance with preferred embodiments, at volumes between about 30 and 150 ul, less than about 500 ul, about 230 ul, and about 100 ul. One embodiment of the present invention is directed to establishing a hysteroscopic insemination 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. Furthermore, the present invention is directed to a hysteroscopic insemination sample comprising a reservoir element a catheter system to which the reservoir element is responsive, and a hysteroscopic compatible volume of sperm contained within said reservoir element. The hysteroscopic compatible volume of s Helm contained within said reservoir element may comprise a volume selected from a group consisting of: between about 30 and 15U ul, less 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, Me sample may be placed within a reservoir elemcut or other sample holding element responsive to the catheter or catheter system. A catheter or cadleter system should tee understood to define any device, system ormethodofinserdonto canals, vessels, passageways, or body cavities to permit injection or Withdrawal of fluids, and in accordance with preferred embodiments such injection or withdraw may provide Me response of the reservoir element or sample holding element, or to keep a passage open.

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 inserniron procedure, as more particularly described below. PreferTcd embodiments may provide the strobing 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 insemination sample may be aspirated into an equine GIFT catheter (2) (Cook Vetennay Products, Brisbane, Australia) using preferably a mL disposable syringe (4) attached to the injection port (6) on the distal end (8) of the catheter. The loaded catheter may be withdrawn into a tube, or preferably an outer polypropylene caDnula (10), 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 We present invention, a determination may be made as to an estrous time for a fetnale mammal. In accordance with an embodiment of the invention, estrus may be induced to determine the estrous time and, for multiple mares, even synchronized. Estrus may be dewed as a state m which the female mammal is capable of conceiving and estrous cycle may be defined as the correlated phenomena of the endocrine and generative systems of a female mammal, potentially Mom the beginrung of one period of esters to not later than the beginning of the next. In accordance with a preferred embodiment, es'aus may be induced, and for multiple female mammals synchroruzed, by administering a substance such as a progestagen, preferitly for mares altrenogest, and preferably lOml orally, potentially for 10 consecutive days, followed by 250 pg cloprostenol i.m., potentially on day 11. Furthermore, a female mammal may be induced into owlation at the time of insemination. Ovulation may be induced, and in preferred embodiments, by the adrninishation of 3000 iu human Chorionic Gonadchopin (#CG, Chorulon, InteIvet, Inc., Millsboro, Holland), preferably admsteredintraYenously at the time oúinsemination or up to approximately 8 hours previously. Ovulation may even be induced, in preferred embodiments, by the adrrunistration of from about 2000 to about 5000 in human Chorionic Gonadotropin.

In preferred embodiments, es$rous cycles may be synchronized by administenog a synthetic progestagen altrenogest (0.044mglkg p.o., llegunate; Hoechst Rousscl Vet, Warren, New Jersey, USA) daily for to consecutive days. Luteolysis may be induced, in preferred embodimentswiththe prostaglandin analogue, cloprostenol (250,ugEstrumate, i.m.; Bayer Corporation, AgGU1tUre])iYiSiOD, Shawnee Mission, Kansas, USA) administered on the eleventh day.

A tLme when the female mammal is appropriately fertile may be determined. The ovaries may be examined, and in preferred embodiments examined ultrasonographically, and preferably every second day until a follicle, and i n accordance with preferred embodiinents a dominant follicle, preferably of> 30mm diameter, is detected. The female may be examined until a follicle, preferably pre-ovulatory, of preferably 35mm is detected. one embodiment, the female may be inseminated during the same day as estrous inducement or synchronization, the same day as ovaiabon inducement or synchronization, or the same day as eslrus and insemination inducement or sychronization.

Next In accordance win particular embodiments of the invention, the Wide element, and in preferred embodiments the optical element (12), may be vagiDlly inserted into the female. Furthermore, the catheter (2), and in preferred embodiments the reservoir element, may be;nserted into the female. The sequence of insertion ofthe optical element(l2) andante caterer (12) may be sequential or coincidental in time.

The guide element or optical element (12) and the catheter (2) may then beguided through the vagina (20) of Me female, as depicted in Figure 2. In accordance with one embodiment, the optical element (12) and catheter (2) may be manually guided. In fact, We catheter (2) may be manually guided without the optical element (12). However, accuracy in finding We l)TJ and the potential result in increased fertility rates, particularly for low numbers of ppam and overall potential efficacy rs We procedure, may require a more accurate guidance procedure. In preferred embodiments, the optical element or endoscope, preferably saleable endoscope, inpreferred embodiments having dimensions of 1.6 m longvithanouter diameteroP12 mm, maybe gliidedthrouthe cervix (22) end propelled forward through the uterine lumen (24), or in additional embodiments, through a uterine hom of We female of the species. An added benefit of We use of the videnendoscope can be lack of a need to rectally guide the insemination process, as may have been required in pest efforts by those of skill in We art.

The uterotubal junction (UIJ) (30) of the female mammal may then be located, preferably optically with the optical element (12). The catheter (2) may then be positioned in the vicinity of or proximate to Me uterotubal junction In accordance with preferred embodiments, the endoscope may be directed under visual control along the uterine horn (26) ipsilateral to the ovary containing the pre-ovulatory follicle (28). Preferably the tip of the endoscope may be directedproximate to, and in preferred embodiment, within about 3-5cm of said uterotubalJunction, andin preferred embodlsnents within about 3-5 cm ofthe papilla (32) of the uterotubal junction.

Atleast a portion ofthe artificial insemination sample may then be exudedfrow the catheter (2). In accordance with preferred embodiments, the outer cannula (10), followed by the irmer GIFT catheter (2) containing the sperm suspension, may be exuded from tile working channel of the endoscope until the tip of the GIFT catheter touches the uterotubal junction, and in preferred embodiments, touches the papilla 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 Me catheter, thereby potentially cteadug an aspirated sample. Such aspirated samples should be understood to include bubbled samples and frothed samples. An aspirated sample may not only provide better adherence to IO a surface in We vicinity of the VTJ, but may finisher 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 with Me sperm cells and to which the sperm cells are responsive. Such alternative I embodiments should tee conuedasdisclosedwithregardto all embodiment ofthepresent invention, either in single orin combination, and shouldbe construed to be disclosed as such.

A low number of sperm may be deposited in the vicious of the uterotubal junction In accordance with preferred embodunents, a plunger ofthe syringe (4) may be depressed to deposit the sample, and in particular embodiments, a small volume (preferably perhaps 1001l1) of Me sample, preferably onto the surface ofthe papilla. The guide element, and in preferred embodiments the optics element (12), and the catheter (2) may be withdrawn from the uterus ofthe female mammal. Inaccordancowithpreferredembodiments, filtered air may be introduced within the uterus of the female to facilitate passage of the instruments tbrou the uterine lumen. Furthermore, in accordance with preferred embodiments, the filtered air may be evacuated from the uterus, preferably simultaneous to the withdrawal ofthe optical element (12) and catheter (2).

The placing of a 1OW nurnJoer of spew may be accomplished, in one preferred embodiment of the invention, by placing with the catheter (2) a number of spew, preferably numbers selected from: less than about tenmillion sperm, fess then about five million spend, less than about two million sperm, less than about one million sperm, less than about five hundred thousand sperm, and less than about one hundred thousand sperm.

Subsequently, the fertilization of an egg of Me female may occur and We production of an offspring mammal from the fertilized egg. In accordance with preferred embodiments of the invention, success levels of fertilization may be statistically comparable to a conventional uterine body acacias insenunation process. Statistically comparable success levels may be defined as for. tilization 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, } O and at least about 90/o of a success rate practically experienced with conventional AI for any particular species. Furthermore, success levels for the present invention may be statistically comparable to a conventional uterine body artificial insemunation process over a sample of cumulative fertilizations whichis greaterthanabout 100, whichis greateran about 500, and which is greater than about 1000. Success rates may fiercer statistically provide for at least a confidence level of at least about 95 percent confidence (potentially expressed as P> or equal to about 0.05), therefore potentially being statistically comparable win conventional AI.

Additionally, success rates for Me present invention may potentially have the same P value oYerarangeofdifferingspermnurnbers,potentially such es 1, 5 or lox 106 sperm for equine.

Furthermore, a success level or rate may be expressed in terms of sample size, whereby the present invention Quay provide, in preferred embodiments, any ofthe aforementioned success rates over a power calculation (it) of at least about 80 percent. Additionally, the success rates, in preferred embodiments, given particular insemination doses for sex-sorted stallion spermatozoa, may every apply to a low number of spermatozoa and mar 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, equids, and swine.

In regard to sperm Viability, longevity and nobility, for example, the present imention may provide for establishing an artificial insemination sample utilizing flesh sperm. The term "fresh sperm" may be broadly defined as sperm that has not been treated, processed or preserved in any manner such that the sperm viability, longevity and/or mobility might be compromised. Such treatment, processing or preserving may include, for example, the sorting of sperm, the freezing and 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 in accordance with embodiments of the present Invention herein may permit the use of low numbers of sperm for insem nation even, for example, when using poor quality collected semen.

Altemative embodiments of the present invention may broadly provide for treatment, processing and preserving of insenunation sperm. Altemative embodiments of the present invention may provide, for example, for the selection of the collected sperm cells more likely to achieve insemination. According to one embodiment of the present invention the 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 centrifilng Me sperm through density gradients, and in preferred embodiments a Percoll gradient. A potentially preferred embodiment may use a gO:45% 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 the use of Percollpre orpostprocessingofthe sperm, and inparticular, potendally pre orpost sorting. Therefore, in accordance with embodiments herein, the Percoll gradient procedure may be used with "fresh sperm." lbe sample established from the desired or selected sperm cells may be used to establish the artificial insemination sample, potentially increasing the fertility rates e, at least in part, to the potentially higher rate of viability of the sample. The Percoll gradient procedure in accordance with t he present invention may be conducted in conjunction with the use of lower numbers of sperm placed within the depositing catheter, as more particularly described supra.

Additionally, establishing an artificial insemination sample may be provided by establishing asamplehavinghysteroscopic compatible media, thereby providing forpotentially increased fertility rates and an efficacious insemination procedure. More particularly, an embodiment of the invertion may provide establishing an artificial insemination sample, and in preferred embodiments having a low number sperm compared to natural insertion doses, and providing for Me establishment of an artificial insemination sample compatible media, for example extender, andinpreferredembodiments, a skim railk medium, suchasEZ Mixin CST (Animal Reproduction Systems, Chino, CA), preferably as a diluting media, potentially prior to additional processing. In accordance with preferred embodiments ofthe presertinventron, dilution may occurtonomoreana2:1 ratio, to no more then aS:1 ratio, and to no more than a 10:1 ratio, to potentially achieve at least a hysteroscopic compatible volume or media with appropriate concentrations. Furthermore, embodiments of Tic present invention may provide fortheuse of an extender, potentially a second extender provided after spermprocessg end potentially inconjunctionwith afirstextender, as previously mentioned, to establish the sample utilizing hysteroscopic compatible media. The second extender may serve to resuspend the sperm sample after processing, and more particularly, provide for a sample utilizing hysteroscopic compatible media. According to preferred embodiments, the medium or second extender may comprise a TALP medium, a HEPES-bufered Tyrode's medium, and an Androhepmedium. In accordancewithpreferred embodiments ofthe present invention, dilutionmay be performed with a skimrniLkmedium such es 3MixinCST4D, win a TALP medium, with aHEPES-buffered Tyrode's medium, and with an Androhep medium, either single or r combination, to potentially achieve at least a hysteroscopic compatible volume or media with appropriate concentrations.

An additional embodiment of the present invention may also provide for an artificial] insemination sample utilizing ahysteroscopic compatible media ormedimahavng a catheter coordinated viscosity. The viscosity may, for example, potentially facilitate the steps of extruding 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 of the blood of said mammal or greater than about Mat of a saline solution. Furthermore additional embodiments may provide for compatible media having a viscosity of Heater than about 1 OOcp, a media having a viscosity of greater than about 300cp, a media having a viscosity of greater than about 1000cp, 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.

Furthermore, an embodiment ofthe present invention may provide a hys; teroscopic insemination sample comprising a reset voir element, a catheter system to which said reservoir element is responsive, apluralky of sperm cells contained within said reservoir element; and bysteroscopic 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 sperm cells as compared to Me number of spenn cells typically poured a natural insemination.

Altering a property of the insemination specimen or sperm 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 then altering a proper of said insemination specimen to establish an artificial insemination sample at about said estrous time. Alternative embodiments may provide altering a property of the 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 proper of the insemination specimen within B time selected from: withm about twenty- four hours of said time determined for so d female of said species of said mammal to be appropriately fertile, within abouttwelve hours of said time deterrninedforsaidLemale of said species of said mammal to be appropriately fertile, within about eight hours of said time determined for said female of said species of said mammal to be appropriately fertile, within about three hours of said brne deterrmned for said female of said species of said mammal to be appropriately fertile, and within about one hour of said time determ ned for said female of said species of said mammal to be appropriately fertile. This alteration may consist of preparing tile sample, sorting the speIm, hawing the sperm, or the like.

One particular embodiment of tile 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 embodiment of the invention may provide preparation of the semen through centrifugation. Me semen may be diluted to provide, for example, 100 x 106 spermatozoa/,nl in preferably a commercial skim milk extender (EZ-IvIi CS1@, Animal: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% Percoll (Sigma Chemical Co., St. Louts, MO, USA) discontinuous density gradient with He goal of reconcentrating the cells and to potentially select a highly motile fraction of spermatozoa The 90% Percoll may be diluted at a ratio of 1:I (rev) with HEPES-buifered Tyrode's medium (Grendahl e' al., 1996) to make a 45% solution In a preferably 15 centnfige tube, preferably 1 mL of 45% Percoll may be carefully layered on top of preferably 1 mL of 90% Percoll. Preferably 1 mL ofthe sperm suspension (100x 106 spenn/mL inEZ-xin, CST)may belayeredontop of the Percoll layers, and the tube may be centrifuged at 800 x g for apreferred period of 12 minutes. Aflcr ccntrifigation, the supernatant may be completely removed andante pelletmay be resuspended in preferably 6001 HEPES-buered Tyrode's Medium. The sperm concentration may be determined, in accordance with one embodiment, using a Densimeter (534B MOD-l, Animal Reproduction Systems, Chino, CA) and the potential volume to deliver 5 million spermatozoa (potentially of a preferred 1001) may be calculated and prepared Or insemination.

In accordance with a broad aspect of He present invention, one embodiment Hereof may provide for the positioning oftbe catheter near the VTJ office female species of mammal whereby the catheter is inserted finder a surface in the vicinity of the uterotubal junction At least a portion of the sample may be extruded or deposited in the vicinity of the UTJ under the surface.

One embodiment of He present invention mar provide for the collecting of sperm cells Tom 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 in a catheter. A determination of a time when He finale is appropriately fertile may be determined, as described above. The optical element (12) and the catheter (2) may be inserted I) 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 inserted under a surface (34) in Me Vicinity of the UTJ (30), as depicted in Figure 3. At leastaportion of smdartificial inserninationsarnplemaybe extruded under the surface Within the Vicinity of the UTI, thus creating a 'blister" with the 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 endomcium or over portion of We uterus or tte uterine lining. ID accordance with one preferred embodiment, the cathetermay be inserted suchthat aparticular portion of the uterus is not pierced, and in accordance win one embodiment, such that a vasculanzed portion of the uterus is not pierced. A vasculanzed portion of the uterus that may not be pierced, according to one particular embodiment, may include, for example, the mesometrium or the myometrium portion of the uterus or other vascularized portions.

Thereafter, the optical element and catheter may be withdTawrt from the female and I 5 fertilization of an egg of the female may occur after which production of an offspring mammal may be expected from the fertilized egg.

An embodiment of We present inventionmayalsoprovide apotentially corresponding insemination catheter having a guide element, or in preferred embodiments a videcendoscope or acaunula, aresenoir element responsive to the guide element, an extrusion element, or in preferred embodiments a synngeto whichsmdreservoir element is responsive, and a cellular piercing tip (363 positioned in front 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 in the vicinity of the tip. One embodiment of an insemination catheter of the present invention is depicted in Figure 3.

As previously described and in accordance with the insemination system of extrusion and deposition under a surface in the vicinity of the UTJ described above, a number of steps of producing a mammal may be perfonned. low number of sperm may be placed In the catheter (2), and preferred embodiments, preferably munbers selected from: less than about ten million sperm, less than about five million sperm, less Man about two million sperm, less I than about one million sperm, less than about five hundred thousand sperm, and less than about one hundred thousand sperm. Furthermore, the fertilization of an egg may be performed in accordance untie the preferred embodiments of file invention wherein success levels success levels of fertilization may be statistically comparable to a conventional uterine body artificial insemination process. Statistically comparable success levels may be defined as previously mentioned.

Furthermore, We sperm cells may be collected from a male species of mammal, in alternative embodiments of the invention, comprising bovlds, squids, or swine. In accordance with alternative embodiments of the invention, spenn cells may be selected from collected cells for those cells mat may be more likely to achieve insemination, as previously described Accordingly, an insemination containment clement may be provided, in accordance with embodunents of the invention, preferably comprising a cellular base surface, and in particular embodiments a uterine lining or, in accordance tenth preferred embodiments, a! nonvasculanzed portion ofthe uterus, such as Me mesomeóium orate myometrium, a cellular cover surface adjacent to the cellular base surface, and in particular embodiments, the endometrium or uterine lining, a substantial enshrouded volume between Me cellular base and Me coyer surface, and in preferred embodiments locatedinthevicimty ofthe UTJ, and spend cells Mom the male of the species. Preferred embodiments may also utilize low numbers of sperm relative to natural insemination, located within the volume and a sperm emission I element adjacent the volume through which spend may pass. The sperm may be collected, selected, of an inseminate volume, perhaps even of an epididyrnis origin, or of any other limitation previously discussed. The sperm emission element may comprise a breach in the endometrium surface of the uterus, as depicted in Figure 3, or may simply occur bar diffusion or the like.

Additionally? other potentially independent procedures may be incorporated into the present inventlor and may still be considered as within the scope Qf Me present invention.

Such procedures may include sorting the spenn cells by a sex characteristic, thereby 21 1 establishing a sex-sorted artificial insemination sample,and inpreferred embodiments having I a low number of sperm compared to a natural insemination dosage for said mammal, may include establishing a low dose sex-selected artificial mseminabon sample. Furthermore, presenting or freezing, and the subsequent Hawing of, sperm sells may be accomplished in particular embodiments, particularly in regard to venous mmrnals such as equid, boYid and swine Depositionoftheinserninationsample'may Reprocessed oraspiratedin any way, may be deposited with the crypts or folds of Me U1 J. and may provide some type of preservation of the sperm for subsequent insemination. Establishment of an insemination specimen or msemmation sample at ahysteroscopic compatible volume and udling compatible mediamay fiercer provide for allowing cooling of the specimen or sample at room temperature.

Centrifugation may preferably beperformedthrough aPercoll gradient for about five minutes at about 200g and for about ten minutes at about 800g. In accordance with a preferred embodiment, concentrating the more motile sperm may be limited to concentrating to less than! about twice ache staring concentration. In particular, the 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 pert! to the producing of an offspring mammal, may further be considered to disclose an embodiment of an animal produced utilizing a process as described in any of the foregoing method claims.

Sorting, in accordance with embodiments of &e present invention, may particularly provide for collecting sperm cells from a male of a species of mammal, sorting the sperm ceils I according to a sex-specific characteristic, establishing a sorted, sex-specific artificial! insemination sample, planing the sorted, sex-specific artificial insemination sample in a catheter; among the various other aspects of &e invention disclosed herein that might be incorporated in method of producing a mammal.

As can be easily understood Tom We foregoing, the basic concepts of the present invention may be embodied in a variety of ways. It involves both insemi Hi on techniques as well as apparatus to accomplish appropriate insemulation. In this application, the inserninion techniques are disclosed as part of the results shown to be achieved by the venous devices 22 1 described and as steps which are inherent to utilization They are simply the natal result of I utilizing the devices as intended and describe 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. Importantly, as to all of the foregoing, all of these facets should be

understood to he 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 Me 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 or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described device-oriented terminology, each element ofthe device implicitly performs a traction. Apparatus claims may not only be included for the devices described, but also method or process claims may be included to address the functions the invention and each element performs. Neither the description nor the terminology is intended to limit the scope of hZe clauns which will be included in a hill patent application.

It should also be understood that a variety of changes may be made without departing from 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 the various elements of the invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such vanation, be it a variation of an embodunent 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 be expressed by equivalent apparatus terms 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 t 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. Similarly, each physical element disclosed should be understood to encompass a disclosure of Me action 3 which that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of an "extruder" shoed be understand to encompass disclosure of the act of "extruding"-whether explicitly discussed or not - and, conversely, were there only i disclosure of the 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, orrules mentionedinthis applicationforpatent, 2 or patents, publications, or over references mentioned in this application for patent, are: hereby incorporated by reference. In addition, as to each tenn 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 lean and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictiormy, second edition are hereby incorporated by reference. However, as to each of the above, to the extent Mat such information or statements incorporated by: reference might be considered in&onsistentwiththe patenting ofthis/these invention(s), such statements are expressly not to be considered as made by the applicant(s).

Thus, the applicant(s) should be understood to have support to clR In at least: i) each oftheinsennationdevices es herein disclosed end described, ii) therelatedmethods disclosed i and described, iii) similar, equivalent, and even implicitvariations of each of these devices and 3 methods, iY) those alternative designs which accomplish each of the functions shown as are 5 disclosed and described, v) those alternative designs and methodswhich accomplish each of 3 the Unctions shown as are implicit to accomplish Mat which is disclosed and described, vi) i each feature, component, and step shown as separate and independent inventions, Yii) the applications enhanced by the various systems or components disclosed, Yiii) the resting products produced by such systems or components, and ix) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying examples, and x) the various combinations andpermutations of each ofthe elements disclosed Further, if or when used, the use of the transitional phrase "composing" is used to maintain the "operand" clams 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 "complying", me intended to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any over 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 Me terrn';at feast one" as usedinthe following description and claims is not intended nor used in this disclosure to mean Mat 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, inherently including devices or methodshaving additional elements or steps apart from those claimed, into "closedended" claims wherem devices or methods having additional elements would not be covered by such claims. Accordingly, if or vhenused, the use of the ansidonal phrase "comprising'' is used to maintain the "open-end" claims herein, according to traditional clann interpretation.

I. PATENT DOCIJMENIS _.

DOCUMENT NO. DATú NAMllCOUNTRY CLASS SUBCLASS Fl[JNG DATE 5,135,759 08104/92 Johnson 424 561 04126/91 1 6,071,689 06/06100 Sadel d e1. 4;5 2 01/29198 I _.

60/238,294 05/10/00 Morris et al. 05/10/00 WO 9834094 061088 NZ. - _ __.. _ 1

_ _ _. .

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11. O 1;R DOCU1\IETS gDCiUding Author, Title' Date, Perffnent Pages, Eta) Bracher, V. and Allen, W.R, 'iVideocudoscopic Examination of the Mare's Uterus: Findings in Normal Fertile Mares", Equine Veterinary Journal, VoL 24 (1992), pp. 274-278

_

Buchanan, B.R, at e1, 'insanloation of Mares with LowNumbers of Either Unsexed or Sexed Spamatozoan, Theiiogenology, Vol. 53, pp 1333-13", (2000)

_

Casliclc, Bay, "late Vulva and the Vulvo-vaginal Orifice and its Relation to Genital becalm of Be Thoroughbred Mare-, Corned Veferinadan, VoL 27, 1937, pp. 178-187 (>an, D.G., et al, Production of Lambs by Low Dose Intrauterine Insernmation with Flow CytonetricaUy Sorted and Unsorted Semen' Theriogenology, Vol. 47, pp. 267, (Abstract), (1991) - , Fugger, EF., "Clinical Bxperiencc with Flow Cytometric Sepalation of Human X- and Y- Chrornosorne Bearing Sperry", Tberiogenology, Vol. 52, pp. 1435-1440 (1999) Grondahl, C, d al, 'on Vitro Production of Equine Embryos' Biology of Reproduction, Monograph Series I, pp. 299-307 (1995)

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Johnson, LN and Welch, G.R, "Sex Presclection: High-specd flow cytometric sorting of X and Y spum for maximum efficiency/', Iheriogenology, Vol. 52' (1999), pp. 1323-1341 Johnson' L. A., "Advances in Gender Preselection in Swine" Journal of Reproduction and Fcrblity Supplanent, Vol. 52,pp. 255-266 (lg97) lohnsor, L.A., Hex Preselection in Swine: Altered Sex Ratios in 0ffspnag Following Surgical Insuninatian of Flow Sorted X- and Y-BearingSperrn", ReproductioTinDonesticAnirnals,Vol. 26,,op. 30314 (1991) . . . Johnson, L A., et al., "Sex Preselection in Rabbits: Live Births from X and Y Sperm Separated by DNA and Cell Sorting" Biology of Reproduction, Vol. 41, pp. 199-203 (1989) _. . Manning S.T., of al., "Devdoprnent of Hysteroscopic Insunination of the Uterine Tube in the Mare", Proceedings of the Annual Meeting of the Society for Theriogenology, 1998, pp. 84 85.

Morris, AH., d al., "Hysteroscopic insemination of reroll numbers of spermatozoa at the uterotub#l junction of preovulatory mares", Journal of Reproduction and Fcrblity, Vol. 118, pp. 95-100 (2000)

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Parrish, J.J., et al., "Capacitation of bovine sperm by heparin", Biology of Rcproductior', Vol. 38, pp. 1171-I 180 (198B) Pcippo, 1., at al., "Sex diagnose of equine Reimplantation embryos using the polymerase chain reaction", lleriogenology,Vol. 4461o271995) Pickett GW, et al., Ydanagernent of the mare for maximum reproductive efficiency" Bulletin No. 6 Colorado State University, Pt. Collins CO. (1989) Schenk J.L., "Cryopreservationofdow-sortedbonnespermatozoa", lheriogenology, Yol.52, 1375-1391(1999) Schoud ILL., et al, "Fertilization with Sexed Equine Spermatozoa Using Intracytoplasmic Spenn Injection and Oviductal Irwannation I, 7th International Syrnpoiun On Equine Reproduction, pp. 139 (Abstract) (1998)

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Seidel, G.E. Jr, et al., "Artificial Insemination of Heifers with Coaled, Unhozen Sexed Scrnen ", Ihenogenology, Voh 49 pp. 365 (Abstract) (1998)

_

Seidel, G.E. Jr, et al., 'seminstion of Heifers with Sexed Sperm ", Theriogenology, Vol. 52, pp. 1407-1421 (I 999) Squire% L, yearly Embryonic Loss" in Equine Diagnostic UKrasonography, 1 Ed. pp 157163 Bds R=teoso ok McKiDnon. Williams and Wilkins, Baltimore, Maryland (1998) Squires, AL., et al, "Cooled and frozen stallion semen", Bulletin No. 9, Colorado State University, Ft. Collins, CO. (1999) - . Vazquez J., et al., "A.I. in Swine; New Strategy for Deep Insemination with Low Number of Spermatozoa Using NDn-surgical Methodology", 14* International Congress on Animal Reproduction, Vol. 2, StDcldlorn, July, 2000, p. 289.

Vazquez, J., et al., "Nonsgicial Utero4ubal lion in the Mare", Proceedings Annual Meeting of the Society for Iheriogenology, 1998, pp. 82-83.

Vazquez, J., et al., "Successful Low-Dose Insemination by a Fiberoptic Endoscopc Technique in the SDW' Proceedings Annual Conference of Be Interatiorml Embryo Transt'er Society, Netherlands, Iheriogenology, Yol. 53, Iouary, 2000, pp 201.

Vazquez, J., et al., "Development of a Non-surgical Deep Ultra Uterine Insennnation Technique", IV intonational Conference on Boor Seined Preservation, Maryland, August, 1999, p 35 and photo of display board. - . .

Vazquez, J., et al., UDevdopucut of a Non-surdeal Deep Intra Uterine Insemination Technique' Boar Semen Preservation IV, IVth Intenahonal Cordercnce on Boar Semen Presavadon, Maryland, pp. 262-263.

Yazquez, J., et al., Iypoosmodc Swelling Test as Predictor of talc Membrane Integrity in Boar Spermatozoa", Boar Semen Preservation IV, IVth International Conference on Boar Semen Preservation, Maryland, pp. 263.

Johnson, L., et d, "Sex Preselechon in Swine: Flow Cytometric Sorting of X- and Y- Chromosome Beanag Sperm to Produce Offspring-, Boar Semen Presentation IV, 2000, pp. 107-114.

Rath, D., et al., "Low Dose Inseminah'on Technique in the Pig' Boar Semen Preservation IV, 2000, pp. 115 - 118.

Lindsey, As, et al., 'ysteroscopic Inserninatlon of Mares with Nonfizen Low-dose Ubsexed or Sex-sorh d Spermatozoa" currently unpublished, pp. 115.

Claims (1)

1. Claims for Divisional 2 In the following claims, the numbering sytecn Tom

   the parent case has been left intact Additions to the claims (if any) are represented by an underline. Deletions hom the clams (if any) are represented by al. 128. An usemmation catheter composing: a a guide element; b. a reservoir element responsive to said guide element; c. an extrusion element to which said reservoir element is responsive; and d. 8 cellular piercing up positioned in front Resaid reservoir element 129. An insemination catheter as described in claim 128 and former condensing a pierce depth control element positioned in the vicinity of said cellular piercing tip. * * *

   309, A method of producing a maal substantially as hereinbefcre described with reference to the accompanying digs.

   310. An insemination containment element substantially as hereinbefore described with reference to the accompanying drainage 311. An inseminadon catheter substantially as hereinbefore described with reference to Me accompanying drawings.

   312. An insemination sample substantially as hereinbefore described with reference to Me accompanying drawings.

   313' A hysteroscopic insenlmation sample substantially as hereinbefore described with reference to Me accompanying drawings. 1b

   314. A mammal produced by a method substantially as hereinbefore described with I reference to the accompanying drawings.

   315. An aniInal produced using a process substantially hereinbefore designed with reference to the accompanying drawings.