GB2399731A - An insemination containment element - Google Patents

Abstract

The present invention provides an insemination containment element for inserting sperm under a surface in the vicinity of the uterotubal junction (30), which insemination containment element comprises a cellular base surface, a cellular cover surface (34) adjacent to said cellular base surface, a substantially enshrouded volume between said cellular base surface and said cellular cover surface and having sperm cells from a male of a species of mammal located within said substantially enshrouded volume and a sperm emission element adjacent said substantially enshrouded volume and through which said sperm cells can pass. The insemination containment element may be employed in mammalian species such as equids, bovides and swine.

Description

3;!,' a 239973 1 l

SYSTEM OF HY STEROSCOPIC INSEMINATION OF MAWS

This application claims the benefit of, and any applicable phority to, U. S. provisional application number 60123 8,294, hereby incorporated by reference.

s TECHNICAI FIELD

isinventionrelates generally to Me field of insemination olmammals. Specifically' it relates to systems to achieve insemination which may be particularly applicable once spend have been treated or processed in some manner, such as sorting the sperm through flow cytometry. More particularly, the invention may relate to systems to achieve insemination, with a low number of spermatozoa as compared to conventional artificial insemination, Trough hysteroscopic inserninationtechniques. The inventionmay be particularly applicable to systems for inseminating equip, bovids and swine. Furthermore, the invention may be particularly applicable to sex selection of offspring in mammals.

BACKGROUND

Artificial insemination (Al), in venous forms, has been used to achieve successfukates of pregnancyandresultingoffspriag inmamrnals. Traditional forms of AIinclude various in vivo technologies such as conventional intrauterine artificial insemination techniques, ormore particularly, bans cervical artificial inseminator. Natural inserunatior doses may typically be large. For example, in some species, natural insemination may involve sperm numbers on Me order of 2-20 x l O9 sperm. In contrast, conventional Al techniques in the same species may require sperm numbers on Me order of 200-500 x I o6 sperm. This can still be viewed as a large number of sperm, especially if Me spend are processed In some manner. Given the traditional requiremcots for high numbers of sperm to achieve acceptable numbers of pregnancies, other insemination techniques have been sought to address devclopin,g breeding technologies Mat may result in a lower sperm count for subsequent insemination. Low dose insemination has been particularly discussed die PCT publication WO 99/33956, enticed "Sex-Specific Insern nation of Mammals With LowNumberof Sperm Cells." Hysteroscopic insemination ho been proposed and to some extent discussed in Morris et al, 2.O(10, hereby incorporated by reference.

- 5;'-' It should be preliminarily noted that all references mentioned in this application, and including additional reference not mentioned, aIc listed at the end ofthis written discussion, all of which should be considered as incorporated bar 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 hens. It can potentially be used for several other species as well. Tcchnologics have been developed to enhance ormodify thepregnancy and resulting offirpnng of ms, particularly with regard to the processing of sperm and insemination tecines One potenffai limiting factor in optimizing the success of artificial insemination, as recognized in Pickett et al., 1989, may be inseminating with low numbers of spermatozoa.

Pickett et al. recognized/hat apotential minimal recommended dose for conventional artificial insemination in the mare may be as high as 500 x 106 progressively motile sperm With low sort rates of around 700 spermatozoa per second in some Sperry sorting technologies, it may take several days to obtainthe recommended dose of spermatozoa for artificial insemination I 5 this, may not only be unpractical, but the viability of the spermatozoa may also be significantly reduced. Low-dose nsenatioa techniques, therefore, may he a desirability to those skilled in the art to potentially reduce the number of spermatozoa needed for acceptable fertility Rites.

Additionally, what might be considered low doses for insemination Inay not typically result in acceptable fertility rates. Fertility rates may be considered acceptable or statistically comparable, for example, if they are acldevable over a high sample size, range or percentage of the fertility rates resulting Tom conventional Al. Low-dose insemination techniques, therefore, may be a desirability to those skilled in the art to potentially reduce the number of spermatozoa needed to maxnize fertility.

Whether in consideration of low-dose insemination or to improve AI fertility rates generally, an additional concern regarding artificial insemination is the efficiency of the procedure as a whole with regard to the resulting numbers of pregnancies. A number of procedural steps may have been used in conversational Al procedures, such as Me synchronization of esters in breeding mares; the preparation of the insemination dose, more particularly the use of extenders for Me dilution (Kenny et al., 1975) and resuspension of spermatozoa(usg TALP or [EPES- buffered Tyrodets Medium, for example), centrifuging the sample through a density or viscosity gradient (using PERCOLL or the like); assessing viability or motility; and the particulars of the insemination procedure. Inserunation procedures have historically included ultrasound and rectally-guided techniques.

Hystcroscopic insemination of mares has also been conducted, as described in the Vasquez et al., 1998 and Maying et al., 1998 references. Only limited success, however, has been demonstrated utilizing the above described procedures in conventional and in the Vasquez et al. and the Manning et aL procedures. In particular, neither reference may have produced statistically comparable conception rates to conventional AI. In particular, the Vasquez et al. and Me Manrung et al. references may have demonstrated conceptionrates of 33% for 3.8 x l o6 spermatozoa and 22% for 1.0 x 1 o6 spermatozoa, respectively, Which may be considered comparable conception rates relative to conventional Al for the species involved. Despite the previous and substantial attempts at producing an efficientprocedure for PI, heretofore, a long felt but unsatisfied need for an efficient procedure for the hysteroscopic insemination of mammals has existed inbreeding technology. 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 insennation has heretofore existed in current breeding technology Furthermore, deep intra uterine insenit1ation of swine has been conducted as described In the 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 10' spermatozoa However, Vasquez ct al., 2000, may have identified the long felt but unsatisfied need for an msemmatiou procedure to address breeding teclmologies utilizing lower spennatozoa numbers for insemination, such as may result from Current Spenn Sorting technoJoes.

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

et al., 2000, and Making et al., 1998, references, those skilled in the art may have failed to address the identified need for proYidiIlg an insemination technique potentially resulting in high feral ity rates mammals Add the resulting lugh 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.

One important procedural step 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, Procedures for the selection of motile spermatozoa may have been conducted with regard to conventional AI, for example in the reference Grondahl et al., 1996 and in hysteroscopic insemination generally, by establishing a dense or viscosity gradient utilizing, for example, PERCOI,L (Sigma Chemical Co., St. Louis, IO) alone oriu combination with other substances. However, heretofore the substantial efforts to hactlonate viable or motile sperm have not particularly addressed the identified needs for an rnsetion technique potentially providing high fertility rates, high fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to He 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 the spermatozoa that may actually reduce the actual number of viable and motile spermatozoa available Tom 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 in the total number of viable or motile spend Tom 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 Irk which nuty require less efforts to obta n a viable, low-dose msenunation sample.

third potential issue with regard to insemination procedure, generally, is the establishment of a compatible volume for the particular insenation technique. One recognized need, as described above, is the desire to potentially provide higherfertility rates.

A second recognized need, also desenbed above, is the ability to use low numbers of spermatozoa to potentially achieve high fertility rates. "e insemination dose volume may be determined by the particular insemination techrique. However, the dose volume may contain a desirable number of spermatozoa to potentially provide [or 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 He insemination technique, to potentially provide acceptable ibrtilily rates.

However, determining a compatible insemination dose volume for aparticulariasem i nation technique, to further potentially provide higher rates of fertility, has potentially not been established forhysteroscopicinsemination, as thereported fertility rates in Vasquezetal, and Manning et al. may demonstrate.

Additionally, and as previously mentioned, the sperm sample may be processed prior to the insemination procedure. Conventional Al, 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 tile inscrnlua':ion procedure itself.

Incompatibility ofthc sample media may result in lower deposition numbers of spennatozoa or dose volume or a lower fertility rate. Furthennore, the mode or form of the deposited insemination dose or the particular method of deposit during Al may further affect the number of deposited spermatozoa available for conception. However, heretofore the substantial efforts directed toward insernmation media may have not particularly addressed the need for providing an insemination technique potentially resulting in high fertility rates, high fertility rates for lower insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insenunation technique.

An additional factor to consider is the timing of insemination. Insemination timing s may be an important factor, for example, to spend viability and longevity and the timing of the estrous cycle of the matinal. Particularly at issue might be the distant location of sperm sample acquisition (i.e the location of the male mammal) and ultimate location of Me ANTI.

Previous efforts may have been made in conventional Al to preserve the sperm sample prior to insemination and to coordinate the insemination with the estrous cycle. however, heretofore the substantial efforts directed to insemination timing may have not particularly addressed the identified Deeds for, and may have even failed to understand the problems of, providing an insemination technique potentially results in high fertilizer rates, high fertility I rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insenunatior techrque.

The source of the sperm sample may also be of importance to the resulting I insemination. Epididynal acquisition ofthe sperm sample (obtaining sperm sample from the epididymis of We testis; du&tules emerging, posteriorly homthe testis that holds spenn during maturation and that forms a tangled mass before uniting into a single coiled duct which is continuous with the vas deferens) may provide someirerentadvantages es to tim ngofthe insemination and viability ofthe sperm. However, heretofore the sulstalal efforts directed to sperm source, viability andinseminabonng may have notfully addressedtheidentified needs for, end may have even failedto understand theproblems of, providing aninsemination technique potentially resulting in high fertility rates, high fertility rates for low insemination dosages, arid insemination techniques to address issues of efficaciousness, particularly with regard to tle hysteroscQpic insemination technique.

DISCIOSIE OF THE INVENTION

The preset inventors have recoded Me potential problems associated with conventional methods of artificial insemination. Accordingly, embodiments of the present 2S invention may provide for the production of a mammal through the use of artificial inserninationthatmayaddress inadequacies of previous insemination techniques and systems.

The invention nary comprise, according to particular embodiments, a method of producing a mammal whereby potentially high fertility rates may be accomplished, ferdli rates which may be statistically compatible with conventional AI results, arid potentially high fertility rates m the use of low spermatozoa doses. More particularly, embodiments of the present invention mar provide for the production of a mammal through the use of hysteroscopic insemination techniques. Additionally, the present invendon may comprise embodiments particularly directed at mamma such as equids, bovids, and swine, among other Gammas. Embodiments of the present invention' therefore, may even be considered development away from previous efforts of artificial insermnation.

()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 I O provide a technique of artificial nserninatiou for mammal production such that lower numbers of spermatozoa may be used in the insemination dose relative to conventional AI and other inseminationtechniques, while, in particular embodiments ofthe invention, at least statistically comparable success rates in fertility are maintained.

A Bother obj ect 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 spenn sorting. A goal of the present invention, therefore, is to provide a technique of artificial insemination HI matinal production tot achieves statistically comparable success rates in fertility, compared to conventional Al and other insemination techniques, with lower-doses of spermatozoa.

Furthermore, an object of the invention is to provide for Me 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 Osseous cycle timing, spennatozoa source, viability, longevity and processing, insemination dose media and volume, arid insemination tunung may be optimized, particularly for low-dose insemination and potentially high fertility.

An additional objectofepresentinvention, according to preferred embodiments, is to provide fortheproduction of a mammal througEhysteroscopic insemination. A goal ofthe present invention, therefore, is to provide a technique of artificial ursemination for mammal production such that the insertion of the insernmAtion 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. l:n accordance win particular embodiments of the invention, blister insemination and bubble or froth insemination may be introduced as preceded embodiments to optimize ferdlib 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 Me insemination dose.

Additionally, an object of the present invention, accordance with particular embodiments, is to provide for the production of various mammal species utilizing an artificial insemination procedure. goal ofthe present invention, therefore, is to provide a technique of artificial insemination for mammals such as equids, bovids and swine, among over species.

A further goal is to provide a technique of artificial inscration for various mamlTal species that additionally provides for 10w numbers of spermatozoa in the inscnnation dose and for potentially high fertility rates, particularly rates that may be statistically comparable to conventional AI.

Other objects ofthe invention are disclosed throughout other areas ofthe specification and clarions. Ifs addidon, Be goals end objecvesD,ay apply eicrm dependent or independent fashion to a variety of other goals and objectives in a variety of embodiments

BRIEF DESCRIPTION OF THE DRAWINGS

Figurc 1 is a partially exploded and perspective diagrammatic view of the optical elemcut, cannula, 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 mnmalian species and, in particular, a depiction of artificial insemination in accordance with one embodiment of the present invention.

Figure 3 is a magnified diagramrnadc view of an enshrouded insemination insertion embodiment of the present invention.

MODE(S) FOR CARRNG OUT TO PRESENT INVENTION Me basic concepts of the invention may be embodied in many different ways. The inventive concept may involve the materials, elements, apparatus, device and methods for the production of a mammal Trough artificial insemination. Furthermore, wlule one preferred I 0 embodiment ofthe invention may be particularly directed to tile production of equids through artificial insemination, the broad concept ofthe invention should be construed as a disclosure of the production of mammals in general, and as indicated, to other mammal species such as bovids and she.

As should be unlerstood, the present invention includes a variety of aspects that may ] 5 be used in various combinations depending upon the application's needs. The invention is intended to encompass a variety of embodunerlts of mammal production and combinations thereof. It involves both methods and devices to accomplish the various aspects explained.

to 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 be understood to be encompassed by this patent both independency and ire combination as set forth in the claims now or Later issued.

Accorrlingly, one embodiment ofthe present invention may provide for Me collection of sperm cells Mom a male of the species of interest. In accordance with one embodimcut, sperm cells are collected from one or more stallions of the equine species. According to this embodiment, semen may be collected, and in preferred cmbodimets, semen may be collected with a commercially available artificial vagina, perhaps from at Icast one stallion of known acceptable fertility. An artificial vagLna such as onemade available by Animal Reproduction Systems may be used with an inline gel filter, and inpreferred embodiments, used on alternate days throughout collection. After collection, the semen may be evaluated for gel-bee 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 bovids, equids or swine. An alternative embodiment of the present invention may provide for the collecting of epididymal spenn cells obtained from the epididymis ofthe testis ofthe male species of the mammal. The alternative embodiment providing [or the use of epididymal sperm may be incorporated with all other disclosed embodiments herein, either in single or in combination.

Furthermore, the present invention provides particular embodiments a hysteroscopic insemination sample comprising areservoir element, acatheter systemto whichthe reservoir element is responsive, and a plurality of epididymal sperm cells contained within the reservoir element.

After sperm collection, an artificial insemination sample may be established for the insemination of the female species. In accordance vets one embodiment of the present invention, the sample may be prepared as villa a low number of sperm camped to a natural insemination dosage for the mammal. The sample may have a low rumbler 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 sorting the sperm for particular sexed ha sperm. In accordance with a preferred embodiment of the invention, the spermatozoa may be stained with Hoechst 33342 and sorted into X and Y chromosome-bearing populations based on DNA content usmg a commercially avallal>le SX MoFlo spcnn sorter.

Additionally, auI artificial insernmation sample may be established at volumes, In accordance with prefened embodiments, at volumes between about 30 and 150 ul, less than about boo al, about 230 ul, and about 100 ul. One embodiment ofthe present invention is directed to establishing a hysteroscopic insemination compatible volume, preferably an insemination sample at a volume selected from a group consisting of; between about 30 and ul, less than about 50O ul, about 230 ul, and about 100 ul. furthermore, the present invention is directed to a b.ysteroscopic 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 spew contained within said reservoir element may comprise avolume selected Tom a group consisting of: between about 30 and 150 ul, less than about 500 ul, about 230 ul, and about 100 ul.

Next, in accordance with preferred embolirnents of the invention, the artificial insemination sample may be placed within a catheter or cathetersystem. In accordance with alternative embodiments, the sample may Replaced wiinareservoir clement or over sample holding element responsive to the catheter or catheter system. A catheter or catheter system IO should be understood to define any device, system ormethod of Luserhoninto canals, vessels, passageways, or body cavities topemut injection or withdrawal of fluids, and in accordance with preferred embodiments such Ejection or withdraw may provide the response of like reservoir element or sample holding element, or to keep a passage open Preferably, Me catheter or catheter system may be used in conjunction with a guide element' and in preferred embodiments an optical elemcut or device, and in preferred embodiments an illumination element, to provide guidance in the artificial inseminatiQu procedure, as more particularly described below. Preferred 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 Vetennary Products, Brisbane, Australia) using preferably a 6-mL disposable syringe (4) attaclcd to the 3njectionport (6) on the distal end (8) ofthe catheter. The loaded catheter may tee withdrawn into a be, or preferably an outer polypropylene camula (10), vouch may be responsive to an optical element (12), and in accordance win preferred embodiments, passed don a working channel of a Pentax EPIvI 3000 videoendoscope (Pentax UK Ltd. Slough, Bucks UK).

Accordingly, an instance, time or duration may be determined in which the artificial insemunation may be appropriate. In accordance with preferred embodiments of the present invention, a determination may be made as to an eshous time for a female 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 defied as a state which the female mammal is capable of conceiving and estops cycle may be defined as the correlated phenomena of the endocrine and generative systems of a female mammal, potentially from the berg of one period of estrus to not later than the beginning of the next. hn accordance with a preferred embodiment, emus may be induced, and for multiple female mammals synchronized, by administering a substance such as aprogestagen, preferably for mares altrenogest, and preferably 10ml orally, potentially for 10 consecutive days, followed by 250 fig cloprostenol i.m., potentially on day I I. Furthermore, a female mam:.nal may be induced Into ovulation et the time Insemination. Ovulationmay be induced, and in preferred embodiments, by the administration of 3000 in human Chorionic Gonadotropin (hCG, Chorulon, InteIvet, inc., Millsboro, Holland), preferably administered intravenously at the time of insemination or up to approximately 8 hours previously. Ovation may even be induced, in preferred embodiments, by the administration of Tom about 2000 to about 5000 in human Chononic Gonadotropin.

En preferred embodiments, estrous cycles may be synchronized by administering a synthetic progestagen altrenogest (0.044mg/kg p.o., Regumate; Hoechst Roussel Vet, Warrens New Jersey, USA) daily for 10 consecutive days. I-uteolysis may be induced, in preferred embodimentswithe prostaglandin analogue, cloprostenol (250pgEstrumate, i. m.; Bayer Corporation, Agriculture Division, Shambles Mission, Kansas, USA) administered on the eleventh day.

time when the feinate mammal is appropriately fertile may be Pretended. The ovaries may be examined, and in preferred embodiments examined ultrasonographically, and preferably every second day until a follicle, and ire accordance with preferred embodiments a dorrunant follicle, preferably of> 30mm diameter, is detected. The female may be examined until a follicle, preferably ple-owlatory, 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 ovation inducement or synchronizahon, or the same day

IZ

as esters and insemination inlucewcut or sychroruzation.

Next, in accordance with particularembadiments ofthc invcution, the guide element, and in preferred embodiments the optical element (12), may be vaginally inserted into the female. Furemore, the catheter (2), and in preferred embodiments the reservoir element, may be inserted into the female. The sequence of insertion of the optical element ( 12) and thecatheter (12) may be sequential or coincidental in time.

lye guide element or optical element (12) and the catheter (2) may then be guided through the vagina (20) of Me female, as depicted Ln Figure 2. In accordance with one embodiment, We optical element (12) and catheter (23 may be manually guided. In [act, the catheter (2) may be manually guided without the optical clement (12). However, accuracy in finding the {JTJ and the potential result in increased fertile rates, particularly for low numbers of spend and overall potential efficacy in the procedure, may require a more accurate guidance procedure. Tnpreferred embodiments, the optical element or endoscope, preferably a flexible endoscope, inpreferred embodiments having dunensions of 1.6 m long with en outer diameter of 12 mm, may be guided through the cervix (22) and propelled forward through the uterine lumen (24), or in additional embodiments, through a uterine horn of Me female ofthe species. An added benefit of the 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 skill in the art.

The uterotubal junction (UIJ) (30) of the female mammal may then be located, preferably, optically with He optical element (12). Me catheter (2) may then be positioned in the vicinity of or proximate to the uterotubal junction. In accordance with preferred embodiments, the endoscopemay be directedunder visual control along theuterine horn (26) Ipsilateral to Me ovaIy containing the pre-ovulatory follicle (28). PreferabI, the tip of the 2S endoscope may be directedproxmate to, arid in preferred embodiments within about 3 Acne 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 extuded from the catheter (2). In accordance with preferred embodunents, the outer cannula (10), followed by the inner GIFT catheter (2) contairung the sperm suspension, may be extruded Dom die working channel of the endoscope until the tip of the GIFT catheter touches the uterotubal junction, 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 Mom We catheter, therebypotentially creating all 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 a surface in Me vicinity of the UTJ, but may fit er allow for improved fertility rates.

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

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

In accordance with preferred embodiments, a plunger ofthe synge (4) may be depressed to deposit the sample, and in particular embodiments, a small volume (preferably perhaps 1 Offal) of the sample, preferably onto the surface ofthe papilla. Ike guide element, and in preferred embodiments the optical element (12), and the catheter (2) may be withdrawn from the uterus ofthe female mammal. In accordance wipreferred embodiments, filtered air may be introduced within the uterus ofthe female to facilitate passage of Me instruments through the uterine lumen. Furthermore, in accordance with preferred embodiments, the filtered air may be evacuated f rom the uterus, preferably simultaneous to Me withdrawal of the optical element (12) and catheter (2) .

The placing of a low ntunber of sperm may be accomplished, in one preferred embodiment of the invention, by placing win the catheter (a) a number of sperm, preferably 3 numbers selected from: less than about ten TuIIion sperm, less trail about f ve million sperm, less than about two million sperm less than about one million sperm, less than about five hundred thousand sperm, and less Man about one hundred thousand sperm.

Subsequently, the fertilization of an egg ofthe female may occur and the production of an offspring mammal from the fertilized egg In accordance with preferred embodiments of the reinvention, SUGcess levels of fertilization may be statistically comparable to a conYentionai uterine body artificial insemination process. Statistically comparable success levels may be defined as fertilization rates of at least about 75% success rates, at least about 65% success rates, at least about 60% SUCGesS 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 AT for any particular species. Furtherrnore7 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 than about 00, which is greater than 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 equal to about O.OS), ikerefore potentiaJlybeing statistically comparable with conventional AI.

Additionally, success rates for Me present invention may potentially have the same P value over a range of differing spend numbers, potentially such as I, 5 or 10 x 1 o6 sperm for e4tune.

Furthennare, a success level or rate may be expressed in terms of sample size, whereby the present inventionmayprovide, in preferred embodiments, any ofthe aforementioned success rates over a power calculation (13) of at least about 80 percent. Additionally, the success rates, in preferred embodiments, given particular insemination doses for sex-sorted stallion spermatozoa, may even apply to a low number of spermatozoa and may routinely produce fertihty rates of at least about 90% of those 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 spcnn viabili ty, longevi ty arid mobility' for example, Me present invention may provide for establishing an artificial insemination sample utilizing flesh sperm. The term "Desh sperm" may be broadly defined as sperm that has not been treated, processed or preserved in any manner such Mat the spenn viability longevity and/or mobility might be comprornsed. Such treatment, processing or preserving may include, for example, the sorting of sperm, We 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 Emanation even, for example, when using poor quality collected semen.

Alternative embodiments of the present invention may broadly provide for treatment, IO processing and preserving of insemination sperm. Altemative embodiments of the present invention may provide, for example, for the selection of the collected sperm cells more likely to achieve insomination. Accordingto one embodirnentofthe present invention the selection of desired Sperry cells or Sperry cells more likely to achieve insemination may comprise concentrating die more motile sperm collected. Additional embodiments may provide the step of centrifuging the sperm through density gradients, and in preferred embodiments a Percoll gradient. potentially preferred embodiment may use a 90:45% Percoll gradient An additional cmbodirnont ray comprise limiting the concentration to less than about twice the starting concentration. [rnplementation of the Percoll gradient should be understood to encompass Me use of Percoll pre or post processing oFthe sperm, and inparticular, potentially pre or post sorting. Therefore, in accordance win embodiments herein, the Percoll gradient procedure may be used with "fresh spenn." lithe sample established from the desired or selected sperm cells may be used to establish the artificial insemination sample, potentially increasing the fertility rates due, at least in part, to the potentially higher rate of viability of the sample. The Percoll gradient procedure in accordance with Me present invention may be conductedinconjuaction with the use of lowernumbers of sperrnplacedwithinthe depositing catheter, as more particularly described supra.

Additionally, establishing an artificial inserration sample may be provided by establishing a sample having hysteroscopic compatible media, thereby providing forpoteritially increased fertility rates and an efficacious insemination procedure. More particularly, an embodiment of the invention may provide establishing an artificial msemmation sample, and in preferred embodiments having a low number sperm compared to natural insemination doses, and providing for Me establishment of an artificial insemination sample compatible media, for example extender, and in preferred embodiments, a szr1 milk medium, such as EZ Toxin CST (Animal Reproduction Systems, Chino, CA), preferably as a diluting media, potentially prior to additional processing. In accordance with preferred embodiments of the present invention, dilution may occur to no more than a 2:1 ratio, to no more than 5:1 ratio, and to no more than a 10:1 ratio, to potentially achieve at least a hysteroscopic compatible volume or media vim appropriate concentradons. F=therrnore, embodiments of the present I O invention may provide for the use of an extender, potentially a second extender provided after spenuprocessing and potentially inconjunctionwth a first extender, as previously mentioned, to establish the sample utilizing hystcroscopic 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 I 5 medium or second extender may comprise a TALP medium, a HEPES-buffered Tyrode's medium, and an Androhep medium. In accordance with preferred embodiments of the present invention, dilution may be performed with a skim milk medium such as 1?%-Mixn CST, with a TALP medium, wiaHI3PES-bufferedTyrode7smedium, am with anAndrohep medit=, either single or in combination, to potentially achieve at least a 1lysteroscopic compatible volume or media with appropriate concentrations.

An additional embodiment of the present invention may also provide for an artificial insenirnation sample utilizgahysteroscopic compatible media ormediumhavg recanter coordinated viscosity. The viscosity may, for example, potentially facilitate the steps of extruding and depositing the sample. Furthermore, in accordance with potentially preferred embodunes of the present invention, the use 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 then about Hat of a saline solution. Furthermore additional embodiments may provide for compatible media having a viscosity of greater than about lOOcp, a media having a viscosity of greater then about 300cp, a mediahaving a viscosity of greater than about 1 OOOcp, a media having a viscosity Ot Mater H= 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 of the present invention may provide a hysteloscopic inserrimation sample comprising areservoirelement, a catheter systemic 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 ate responsive. In accordance with additional embodiments, an insemination containmert whereinsaidplurity of sperm cells are contained within said reservoirelement may include a low number of sperm cells as compared to the number of sperm cells typically found in a natural insemination.

Altering a property of the insemination specimen or sperm cell sample may be conducted accordingtothepresentinventtor, and in accordance with preferred embodiments, determining an estrous tLmc for a female of a species of said manunal and there altering a property of said insemination specimen to establish art artificial insemination sample at about said estrous time. Alternative embodiments may provide altenugaproperty ofthe sample at about the tirade 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 Dom: within about twenty-four hours of said tires determined for said female of said species of said mammal to be appropriately fertile, vvithinabouttwelve hours of said time determined for said female ofsaid species of said mammal to be appropriately fertile, within about eight hours of said time determined for said female of said species of said marginal to be appropriately fertile, within about three hours of said time detemuned for said female of said species of said mammal to be appropriately fertile, and within about one hour of said time determined for stud female of said species of said mammal to be appropriately fertile. This alteration may consist of prepannU the sample, sorting the sperm, thawLng the sperm, or We like.

One particular embodiment of the present invention may provide for establishing a hystcroscopic compatible media and Me concentration of sperm to select sperm more lilcely to achieve insemination. Particularly, one potentially preferred embodiment of the invention may provide preparation ofthe semen through cenhifigation. The semen may be diluted to provide, for example, 100 x 106 spermatozoa/ml in preferably a commercial skim milk extender (E2;- CST, Animal Reproduction Systems, Chino, CA). The sperm suspension may be protected Tom 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. Louis, MO, USA) discontinuous density gradient with the goal of reconcentrating the cells end to potentially select a highly motile Faction of spermatozoa. The 90% Percoll may be diluted at a ratio of l:1 (vlY) With HEPES-louered Tyrode's medium (Grandam et al., 1996) to malce a 45% solution. In a preferably 1 5-mL centrifige tube, preferably 1 rnL of 45% Percoll may be carefully layered on top of preferably 1 mL of 90% Percoll. Preferably I mL of the spenn suspension (I 00 x I o6 spermJmL in E2:-Mixin, C8l) may be layered on top of the Pcmoll layers, and the tube may be centrifuged at 800 x g for apreferred period of 12 minutes. After centiifirgation, the supernatant may be completely removed and Me pellet may be resuspended in preferably 600,u] HEPl:,S-buffered Tyrode's Medium. The sperm concentration may be deterrnined7 in accordance with one embodiment, using a Densimeter (534B MOV-1, Animal Reproduction Systems, Chino, CA) and Me potential volume to dclivcr 5 million spermatozoa (potentially of a preferred 100ll1) may be calculated and prepared for insemination.

In accordance with a broad aspect ofthe present invention, one embodiment thereof may provide for the positioning of the catheter near die UTJ of the female species of mammal whereby Tic catheter is inserted under a surface In the vicinity of the uterotubal junction. At least a portion of Me sample may be extruded or deposited in the vicinity of the I1J under the surface.

One embodimentofthepresentinventionmayprovidefor the collecting of spermcelis from the male species of mammal, establishing an artificial Insemination sample utilizing at least some of the spend cells collected, and placing the artificial insemination sample use a catheter. A deterrninabon of a time when the female is appropriately fertile may be determined, as described above. The optical element ( 12} and He catheter (2) may be inserted vaginally and guided through the vagina, as depicted un Figure 2. Ibe UTIrnay be optically located with optical element (12).

The catheter may be inserted under a surface (34) in Me vicinity of the UTJ (30), as depicted in Figure 3. Atleastaportion of said artificial insemination sample maybe extruded under We surface within the Vigor of the J, Bus 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 ANTI. The surface (34) may comprise the endomeium or other portion of the uterus or the uterine lining. In accordance with one preferred embodiment, the catheter may be inserted such that a particular portion of the uterus is not pierced, and in accordance with one embodiment, such that a vasculzed portion of the uterus is not pierced. A Yascularized portion of the uterus Mat 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 withdrawn from the female and fertilizationofanegg of Me female may occur after which production of an ospang mammal may be expected Mom the fertilized egg.

An embodiment ofthepresent Lnventionmayalsoprovide apote'tially corresponding inseminationcatheterhaving a guide element, orinpreferred embodiments avidecendoscope or a cannula, a reservoir 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 in front of the reservoir element. The catheter may farther provide a pierce depth control element, such as an adjustment element or a stop on the piecing tip (36) positioned in the vicirnty 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 Ln Me vicinity of the UTJ described above, a number of steps cf prmlucing a maternal may be performed. A low number of sperm may be placed in the catheter (2), and in preferred embodiments, preferably numbers selected from: less Man about ten million sperm, less than about five million sperm, less than about two million sperm, less than about one million seems, less than about five hundred thousand sperm, and less than about one hundred thousand sperm. Furthermore, the fertilization of an egg may be performed accordance with the preferred embodiments of the invention wherein success levels success levels offertilization may be statistically comparable to a conventional uterine body artificial insemination process. Statistically comparable success levels may be defined as pTcVioUSly mentioned.

Furthermore, the sperm cells may be collected Dom a male species of mammal, in alternative embodiments ofthe invention, comprising bodies, equids, or swine. In accordance I O with alternative embodiments of the invention, sperm cells may be selected from collected cells for those cells that may be more likely to achieve inseminati on, as previously described.

Accordingly, an insemination containment element may be provided, in accordance with embodiments of the invention, preferably comprising a cellular base surface, and in particular embodiments a uterine lining or, in accordance with preferred embodiments' a I 5 nonvascularized portion ofthe uterus, such as the mesomeium or Me myometrium, a cellular cover surface adjacent to the cellular base surface, and in particular embodiments, the endometriLm or uterLne lining, substantial enshrouded volume between Me cellular base and We cover surface, and in preferred embodnents located in the vicinity ofthe U N. and sperm cells Dom the male of the species. Preferred embodiments may also utilize low numbers of sperm relative to natural insemination, located mtliin the volume and a sperm emission element adjacent the volume through which sperm may pass. Me sperm may be collected, selected, of an inseminate volume, perhaps even of an epididymis origin, or of any other limitation previously discussed. The sperm emission element may compn,e breach En the endometrium surface of the uterus, as depicted Figure 3, or may simply occur by diffusion or the like.

Additionally, other potentially independent procedures may be incorporated into the present invention and may still be considered as within the scope of the present invention.

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 sperm compared to a natural insemination dosage for said mammal, may include establishing low dose sex-selected artificial insemination sample. Furthermore, preserving or freezing, and Me subsequent thawing of, sperm sells may be accomplished in particular embodiments, particularly in regard to various mammals such as equid, bond and swine. Deposition ofthe insemination sample, may Reprocessed oraspirated In any way, may be deposited with the crypts or folds of the I1J, and may provide some t,Ype of preservation of We sperm for subsequent insemination. Establishment of an insemination specimen or insemination sample at ahysteroscopic compatible volume and uti inns compatible mediamay further provide for allowing cooling of the specunen or sample at room temperature Centrifugation may preferably beperformedthrough aPercoll gradient for about five mmutes at about 200g and for about ten minutes at about 800g. In accordance with a preferred embodiment, concentrating the more motile sperm may toe Sited to concentrating to less than about twice Me stag concentration. In particular, the broad and narrow concepts embodied in the present invention should be consumed as applying to over species of mat, 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 forego method claims.

Sorting, lo accordance win embodiments ofthe present invention, may particularly provide for collecting spenn cells from analeofa species of mammal, sorting the spelmcells according to a sex-specific characteristic, establisldrg a sorted, sex-specific artificial insemination sample, placing the sorted, sex-specific artificial insemination sample In a catheter; among the various other aspects of the invention disclosed herein that might be incorporated in method of producing a mammal.

As can be easily understood from the foregoing, the basic concepts of the present invention may be embodied in a variety of ways. It involves both insemination techniques as well as apparatus to accomplish appropriate inseniinabon. In this application, Me insemination techniques are disclosed as part of the results shown to be achieved by the various 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. Importantly, as to all of Me 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 descnptio The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not fully explain the generic nature ofthe invention and may not explicitly showhow each feature or element can actually he representative of a broader function or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Wherc the irvendon is described In device-oriented terminology, each element of the device implicitly performs a fimction. 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 the clauns which will be included in a Fill patent application.

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

They still fall within the scope of this invention.

Further, each of We venous elements of the 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 embodunent, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood Mat as the disclosure relates to elements of the invention, the words for each element may lee expressed by equivalent apparatus teens ox method teams -- even if only the function or result is the same. Such equivalent, broader, or even more generic teens 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 Mat 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 the action which that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of an "extenders' should be understood to encompass disclosure of He act of "extruding" -- whether explicitly discussed or not and, conversely, were there only disclosure of the act of "extruding", such a disclosure should be understood to encompass disclosure of an "extruder" and even a "means for extruding". Such changes and alternative tempts are to be understood to be explicitly included in the description.

Any acts of law, statutes, regulations, or rules mentionedinthis application forpatent, or patents, publications, or other references mentioned in this application for patent, are hereby incorporated by rcDerence. In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such intepretatioz, common dictionary definitions should be understood as incorporated for each telm and all deftiorts, 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 the above, to the extent that such information or statements incorporated by reference might be considered inconsistent Vying tile patenting of islese invention(s), such statements arc expressly not to be considered as made by the applicant(s).

Thus, the applicant(s) should be understood to have support to claim at least: i) each oftheinsemination devices ashereindisclosed and dcsclibed' if) the relatedmethods disclosed and described, iii) similar, equivalent,and even implicitvariations of eachofthese devices an<1 methods, iv) those alternative designs which accomplish each of the Unctions shown as are disclosed and described, v) osc alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish that which is disclosed and described, vi3 each feature, component, and step shown as separate arid independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii3 the resulting products produced by such systems or components, and x) methods and apparatuses substantially as described hereinbefore and with reference to any of the accompanying 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 maintain the "open-end" claims herein, according to traditional claim interpretation. Thus, unless the context requires otherwise, it should be understood that the term "compnse" or variations such as "comprises" or "compnsing", are intended to 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 their most expansive form so as to afford the applicant the broadest coverage legally permissible.

It should also be noted that the term 'fat least one,' as used in the following description and claims is not irteuded nor used in this disclosure to mean that other claims or descriptions not incorporating the "at least one" language cannot farther 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" clauns wherein devices or methods having 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 claim interpretation.

I. PATENT DOCUINTS DOCUMENT NO. DATE NAMEJCOUNTRY CLASS SUBCLASS FILING DATE I 5,13 5,759 08/04/92 hhnson 424 561 04/26/91 _ _ _. _ _ 6,071,689 06106/00 Scidel et al. 4;5 2 0129/g8 1 _.

60/238,294 05/10/00 Moms et al. 05/10/00 WO 98/34094 06/08198 NZ 1 _.

_. . . _ __ _. . , _ II. OTHER DOCUTS (Indudlng Author, Tide' Date, Pertinent Page,, Eta) Bracher, V. and Allen, W.R, 'Videocadorcopic BxaTnination of the Mare's Uterus: Findings in Normal Fertile Mares", Equine Voterilary Journal, Vol. 24 (1992), W. 274-2?8 _.. ...

Buebanan, B.K, at al, 'qnscrnination of Mares with Low Numbers of Eidacr Unsexed or Sexed Spermatozoa", lberiogenology, Vol. 53, Up 1333-1344, (Z000) Caslick, B.A., Arc Vulva and the Vulvo-vagmal Orifice and its Relation to Genital Health of the Thoroughbred Mare", Cornell Vctermarian, VoL 27, 1937, pp. 178-87 (an, D.G., et al, "Production of Lambs by Low Dose Intrauterine Iasernmatior, with Flow Cyto'nctrically Sorted and Unsorted Semen' Theriogenology, Vol. 47, pp. 267, (Abstract), (1997) _.

I O Fugger, IS.F., "Clinical cperiencc with Flow Cytometric Scpaon of Human X- and Y- Chromosornc Beanag Sperm", Theriogenology, Vol. 52, pp. 1435-1440 (1999)

_

Grondahl, C, et al, 'A Vitro Production of Equme embryos", Biology of Reproducunn, Monograph Series 1, pp. 299-307 (1995) Johnson, L.A. and Welch, (}.R., USex P'reselection; High- spced flow cytometric sorting of X and Y sperm for maximum efficiency'', lllenogenology, Vol. 52, (!999), pp. 1323-1341 Johnson' L..A., "Advances in Gender Presebetion in Swine" Joumal of Reproduction and Fertility Supplement, Vol. 52,pp. 255-266 (1997) Johnson, L..A., "Sex Preselechon in Swine: Altered Sex Ratios in Offspring Following Surgical Insemination of Flow Sorted X- and Y-BeanugSperrn", Reproduction m Domestic Animals,Vol. 26,pp. 309-314 (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., et al., "Development of Hysieroscopic Insemination of the Utcrinc Tube in the Mares, Proceedings of the Annual Meeting of the Society for lbenogenology, 1998, pp. 84-85.

Morris, L.H., ct al., "Hysteroscopic insemination of small numbers of spematowa at the uterotubal junction of preovulatory mares", Journal of Reproduction end Fertility, Vol. 118, pp. 9S-100 (2000) Parrish, J.J., at al., 'capacitation of bovine sperm by heptuin' Biology of Reproduction, Vol. 38, pp. 1171-1180 (1988) _. . Peippo, J., et al., "Sex diagnosis of equine prcimplantation embryos using He polyrnerase chain reaction", Theriogenology, Vol. 44 619-627 (1995) Pickett GW, et al., "Management of the mare for rnexirnurn reproductive efficiency" Bulletin No. 6 Colorado State University, Pt: Collins CO. (1989) Schenk, J.L., "Cryopreservationofflow-sortedbovinespermatowa", Theiogalology, Voh52'l3i5-l39ll999)

_

Schmid ILL., et al, 'fertilization with Sexed Equine Spennatozoa Using Intracytoplasrnic Spend Injection and Oviductal Insemination '3 7th International Symposium On Equine R:production, pp. 139 (Abstract) (1998) 3 5 Scidd, (3.E. Jr, et al., "Artificial Insurunation of Heifers with Cooled, Unhozen Sexed Screen ", Iheriogenology, Vol. 49 pp. 365 (Abstract) (1998) Seidei G. E Jr et al 'seminetion of Heifers with Sexed Spenn ", Theriogenology, Vol. 52, pp. 1407-1421 (1999)

--

Sqlures, L, 44Barlynbryosic Loss" in Bquine Diaostic UItraSOnaBIaPhYJ 1 Et. pp 1571631 Rantanen & McK;unon. Williams and Wilkins, Beltimore, Maryland (1998)

__

Squires, B.L.., et al, "Cooled and frozen stallion semen", Bulletin No. 9, Colorado State University, Pt. Collins, CO. (1, 99) Vuzquez, J., at al., "A.I. in Swine; New Sbatc6y for Deep Irsanination with Low Number of Spermatozoa Using a Non-surgkal Medatology", 14. Intemational Congress on Animal Reproduction, Vol. 2, Stockilom, July, 2000, p 289.

Vazquez, J., et al., 'Nonsurgicial Utero-tubal senunatian in the Mare", Proceedings Annual Meedog of Me Society for Theriogenology,1998,pp. 82-83.

_

Prouedlngs Annual Conference once Intematonal Eimbryo Trsusfa Society, Netherlands, ibenogenology, Yol. 53, January, 2000, pp. 201.

-

Vazquez, J., et al., evdoprnent of a Non-surglcal Deep Intra Uterine Inserrination Technique", IV International Conference on Boar Serpent Preservton, Maryland, August, 1999, p 35 end photo of display board. _.

Vazquez, J., et al., "Developtnent of a Non-surgical Deep Intra Uterine InseDination Technique", Boar Semen Presentation IV, IVth International Conference on Boar Semen PresvatioD, Masyland, pp. 262-263.

Yazquez, J., et al., 'Hypoosmotic Swelling Test as Predictor of tile Membrane Integrity in Boar Spermatozoa", Boar Semen Preservation IV, IVY Jntemational Conference on Boar Scrnen Preservation, Maryland, pp. 263.

Johnson, L., et al, "Sex Presclection in Swine: Flow Cytomdnc Sorting of X- and Ye romoswne Bearing Sperm to Produce Offi;pang", Boar Seroe4 Presenation IV, 2000, pp. 107-114.

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

Lindsey, A., et al., 'Hysteoscopic Inseninatton of Mares with NonOozen Low-dose Unsexed or Scx-sorted Spermatowa", curruttlyunpublLshed, pp. 115. t

Claims (1)

1. Claims for Divisional 1 In the following claims, the nnbering system front

   the parent case has been leR intact.

   Additions to the claims (if any) are represented by an underline. Deletions Tom the clauns (if any) are represented by a 6.

   113. An insemination containment element comprising: a cellular base surface; b. a cellular cover surface adjacent to said cellular base surface; c. a substantially enshrouded volume between said cellular base surface and said cellular cover surface; d. sperm cells from a male of a species of mammal located within said substantially enshrouded volume; and e. a sperm emission element adjacent said substantially enshroud volume and through which said sperm cells can pass.

   114. An Insennnation containment element as described an cluing 113 wherein said sperm cells front a male of a species of Cal Iwated within said substantually enshrouded volume comprise a low number of sperm cells as compared to a the nwnb" of sperm cells typically found n a natural insemination.

   115. An insemination containment element as described in claim 114 wherein said low number of sperm cells as compared to a the number of spenn cells typically found in a natural insemluation composes a number of spelln cells selected from a group consisting of: less than about ten million spenn, less than about five million sperm, less than about two million sperm less than about one million sperm, less Han about five hundred thousand sperm, and less than about one hundred thousand sperm.

   116. An insemination containment element as described in claim 115 wherein said sperm cells compuse spenn cells from a male of a species of a mammal selected fiom a group consisting of: bovids, squids, or swine.

   117. An insenunation containment element as described in claim 113 Veered said sperm cells Tom a male of a species of mammal located within said substantially enshrouded volume comprise sperm cells selected Mom sperm cells collected which are more likely to achieve insemination.

   118. An mseminalion contaimnent element as described in claim 117 wherein said sperm cells selected from sperm cells collected which are more likely to achieve insemination comprise sperm cells selected to be Me more noodle spenn collected from said male species of said mammal.

   119. An Patios containment clement as described in claim 113 Herein said sperm cells from a male of a species of mammal located within said substantially encbrouded retune comprise Mesh sperm cells.

   120. An insemination continent element as described in claim 113 vvLerein said sperm cells fiom a male of a species of mammal located within said substantially enshrouded volume has an inseminate volume and wherein said inseminate volume composes a volume selected Bom a group consisting of: between about and lSO us, less than about 500 ul, about 230 ul, and about 100 ul.

   121. An insemination containment element as described in claim 113 wherein said sperm cells from a male of a species of mammal located within said substantially enshrouded volume comprise epididymal sperm cells from a male of a species of mamma.

   122. An insemination containment element as describedin claim 115 wherein said sperm cells Mom a male of a species of mammal located within said substantially enshrouded volume comprise sex-selected sperm cells from a male of a species of Ad.

   123. An insemination core lament element as described in claim 122 wherein said sex- selected sperm cells fiom a male of a species of mammal comprise sperm cells from Me of a species of a mammal selected In a grout> consisting of: bovids, squids, or swine.

   124. An Nation containment element so described in claim 115 wherein said substantia11y enshrouded volume between said cellular tease surface and said cellular cover surfi'ceis located in the vicinity of a uterotubal junction within a female of said species of said mammal.

   125. An usHon containment element as described in claim 124 wherein said cellular corer surcc adjacent to said cellular base surface comprises an endomibimD surface of a uterus within said female of said species of said malDmal - 126. An insemination un,intnent element as described in claim 125 wherein said cellular baso surlicecopuses a vascularized portion of said uterus of said Remade of said species of said may 127. An insemination containment element as described in clailD 115 wherein said sperm emission element adjacent said substantially enshrouded volume and through which said swam cells can pass comprises a breach in said endomitrium surface of said uterus within said female of said species of said mammal. * * *

   309. A method of producing a mammal substantially as hereinbefore described with.

   reference to Me accompanying drawings.

   310. An Insemmation containment element substantially as hereinbefore described with reference to the accompanying drawings. So

   311. An insemination catheter substantially as hereinbefore described win reference to the accompany drawings.

   312. An insemination sample substantially as herenbefore described with reference to the accompanying drawings.

   313. A hysteroscopic mseminado sandpile substantial!,' as herembefore described with reference to the accompanying drawings 314. A mammal produced by a method substantially as heranbefore described with reference to the accompanying drawings.

   315. An anneal produced using a process substaTRBy as heeinbefore described with reference to the accompanying drawings. 3\