GB2399272A - An insemination sample - Google Patents

Abstract

The present invention provides an insemination sample comprising a plurality of sperm cells contained within a reservoir element of a catheter (2), wherein said sample is configured to be positioned in the vicinity of a uterotubal junction (3) of a female of a species of mammal and deposited within the vicinity of the uterotubal junction (30) within said female of said species of said mammal by visually locating the uterotubal junction (30). The insemination sample of the invention may be used to artificially inseminate such mammalian species as equids, bovides and swine.

Description

pt pro': :' ' SYSTEM OF lIYSTEROSCOPIC INSEMINATION OF AS This application

claims the benefit of, and any applicable priority to, U.S. provisional application number 601238,294, hereby incorporated by reference.

TECHNICAL FIELD

This inventionrelates generally to the field of insemination of mammals. Specifically, it relates to systems to achieve insemination which may be particularly applicable once sperm have been treated or processed in some manner, such as sorting the sperm through flow cytometry. More particularly, the invention may relate to systems to achieve insemination, with a low number of spermatozoa as compared to conventional artical insemination, Trough hYsteroscopic inseninationtechniques. The inventionmay be particularly applicable to systems for inseminating equids, bovids and swine. Furthermore, the invention may be particularly applicable to sex selection of of Spring in mammals.

BACKGROUND

Arcial insemination (AI), in venous forms, has been used to achieve successful rates of pregnancy and resulting offspring in mammals. Traditional forms of AI include various in vivo technologies such as conventional intrauterine artificial insemination techniques, ormore particularly, bans cervical artificial msenation. 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 IO9 sperm. In contrast, conventional Al techniques in the same species may require sperm numbers on the order of200-500 x 106 sperm. This can sell be viewed es 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 renumbers of pregnancies, other insemination techniques have been sought to address developing breeding zip technologies Hat 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-Specific InseminabonofMammals WithLowNumberofSperm Cells." Hysteroscopic insemination has been proposed and to some extent discussed in Molris et al., 2000, hereby incorporated by reference.

4 A: : : : :: :' It should be preliminarily noted that all references mentioned in 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.

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 has. It can potentially be used for several other species as well. Technologies have been developed to enhance or modify the pregnancy and resulting of Wiping of rnmmals, pcully win regard to the processing of spenn and insemination techniques.

One potential limiting Actor in opmizg the success of artificial insemination, as recognized in Prckett et al., 19891 may be msemunating with low numbers of spermatozoa.

Pickett et al. recognized that apotential minimal recommended dose for conventional artificial insemination in Me mare may be as high as 500 x 106 progressively motile sperm. With low sort rates of aroma D permatoa per second in some sperm sorting technologies, it may take several days to obtain the recommended dose of spennatozoa for artificial insemination.

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

Additionally, what night be considered low doses for insemination may not typically result in acceptable fertility rates. Fertility rates may be considacd acceptable or statistically comparable, for example, if they are achievable over a him sample size, range or percentage of the fertility rates resulting from conventional AI. Lowose insemination techniques, therefore, may be a desirability to those skilled in the art to potentially reduce the number of spermatozoa needed to maximize 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 I.. ^i.. . - ,,'

procedural steps may have been used in conventional Al procedures, such as the synchrorntion of esters breeding mares; the preparation of the insemination dose, more particularly the use of extenders for the dilution (Kenny et al., 1975) and resuspension of spetmatozoa(usg TALP or HEPE;S-bufFered Tyrode's Medium, for example), centrifuging Me sample through a density or viscosity gradient (using PERCOLL or the like); assessing viability or motility; and the particulars of the insemination procedure. Lusermnation procedures have historically included ultrasound and rectallJ,-guided techniques.

Esteroscopic insemination olmares has also been conducted, as described in the Yasquez et al., Ig98 and Mrung et al., 19g8 references. Only limited success, however, has been demonstrated in utilizing the above described procedures in conventional Al and in the Vasquez et al. and the Meg et al. procedures. In particular, neither reference may have produced statistically comparable conception rates to conventional AI. In particular, the Vasquez et al. and the Maurung et al. references may have demonstrated conception rates of 33%for3.8 x 106 spermatozoa and 22% for 1.0 x 105 spermatozoa, respectively, which may be cansired aon-compale conception-rates relative to conventional Al for the species involved. Despite the previous and substantial attempts at producing an efficient procedure for AI, heretofore, a long felt but unsatisfied need for an efficient procedure for the hysteroscopic insernmation of mammals has existed in breeding 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 insemination has heretofore existed in current breeding technology.

Furthermore, deep intra uterine insemination of some 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 Me order of 20 x 10' spermatozoa However, Vasquez et al., 2000, may have identified the long felt but unsatisfied need for an insemination procedure to address breeding technologies utilizing lower spermatozoa numbers for insemination, such as may result from current sperm sorting technologies.

Therefore, as may have been demonstrated from the Vasquez et al., 1998, Vasquez . . ; I:. ; ,.>,: et al., 2000, and Manning et al., l9Y8, references, those skilled in the art may have failed to address the identified need for providing an insemination technique potentially resulting in high fertility rates in mammals and the resulting high production m mammals, hi&in fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly win 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 msemiration generally, by establishing a density or viscosity gradient utile, for example, PERCOLL (Sigma Chemical Co., St. Louis, MO) alone orin combination with other substances. However, heretofore Me substantial efforts to fractionate viable or motile sperm have not particularly addra,sed the identified needs for an insemination tecbmique potentially providing 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.

A second potential issue with regard to insemuatinn 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 from a particular sample. Heretofore, the substantial efforts to fractionate viable or motile sperm 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 m the total number of viable or motile sperm Tom a particular inseminate sample, previous attempts incorporating a density or viscosity gradient mar have actually taught away from the present procedure of providing for an insemination technique which may require less efforts to obtain a viable, low-dose insemination sample.

A third potential issue with regard to insemination procedure, generally, is the establisbrnent of a compatible volume for the particular insemination technique. One recognized need, as described above, is the desire to potentiallyprovide higher fertility rates.

A second recognized Deed, also desenbed above, is the ability to use low numbers of sperúnatozoato potentially achieve high fertility rates. The msennadon dose volume may be determined by the particular insenunation technique. However, the dose volume may contam 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 He insemination technique, to potentially provide acceptable fertility rates.

However, determining a compatible insemination dose volume for aparcarinsern nation technique, to further potentially provide higher rates of fertility, has potentially not been established for hysteroscopic insenunation, as theTeponed fertility rates in Vasquez et al. and Manning et al. may demonstrate.

Additionally, and as previously mentioned, He sperm sample may be processed prior to the inscrnmation procedure. Convoutional A1, for example, may provide for the use of extenders for the dilution (Kenny et al., 1915) arid resuspension of spermatozoa. However, the particular media used may not be compatible with tle inseminadon procedure itself.

Incompatibility ofthe 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 insemination close ortheparticularmethod of deposit curing Al may further effect the number of deposited spermatozoa available for conception. However, heretofore the substantial efforts directed toward inserrunation media may have not particularly addressed He need for providing an insemination technique potentially resulting in high fertility rates, high fertility rates for low insemination dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insemination technique.

An additional factor to consider is He timing of insemination. Insemination timing s :. t, c,

may be an important factor, for example, to sperm Viability and longevity and the timing of the estrous cycle of the mammal. Particularly at issue might be the distant location of sperm sample acquisition (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. However, 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 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.

The source of the sperm sample may also be of importance to Me resulting insemination. Epididymal acquisition ofthe sperm sample (obtaining sperm sample bomthe epididymis ofthe testis; ductules emerging posteriorly fi om thetestis thatholds sperm during maturation and that forms a tangled mass before unating into a single coiled duct which is continuous with the vas deferens) may provide some irreverent advantages as to timing ofthe insenunation and viability ofthe sperm. However, heretofore the substantial efforts directed to sperm source, viability and insenunabon tinning may have not Filly addressed the identified Leeds for, end may have even failedto understandtheproblems of, providingaIlinsunation technique potentially resulting in high fertility rates, high fertility rates for low nseminadon dosages, and insemination techniques to address issues of efficaciousness, particularly with regard to the hysteroscopic insemination technique.

DISCLOSURE OF T IRON

The present inventors have recognized the potential problems associated win conventional methods of artificial mseminadon. Accordingly, embodiments of the present invention may provide for the production of a mammal through the use of artificial nsenationthatmayaddrcss inadequacies of previous inseminationtechniques end systems.

I he invention may comprise, according to particular embodiments, a method of producing a mammal whereby potentially high fertility rates may be accomplished, fertility rates which may be statistically compatible with conventional AI results, and potentially high fertility rates win the use of low spermatozoa doses. More particularly, embodiments ofthe present invention roan provide for the production of a mammal through the use of hysteroscopic insemination techniques. Additionally, the present invention may comprise embodiments particularly directed atmammals such as equids, bovids, and swine, among othermammals. EmbodiT rents ofthe present invention, therefore, may even be considered development away from previous efforts of artificial Insemination.

One object of the present invention, therefore, is to provide for the production of a mammal utilizing an efficacious procedure. Therefore, a goal ofthe present invention is to provide a technique of artificial insenunationformammalproductionsuchthatlowernurnbers of spermatozoa may be used in We insemination dose relative to conventional AI and other insemination techniques, while, in particular embodiments of the invention, at least statistically comparable success rates in fertilizer 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 Dom breeding technologies such as spenn sorting. A goal of the present invention, therefore, is to provide a technique of artificial insemination for mammal production that achieves statistically comparable success rates in fertility, compared to conventional AI and other insemination techniques, with lower-doses of spermatozoa.

Furthermore, an obj ect of the invention is to provide for the production of a mammal utilizing an artificial insemination procedure that may enhance steps involved in the artificial msemiTtion. To this end, one goal of the present invention is to provide a technique of artificial insenunation for mammal production such that steps of cstrous cycle timing, spennatozoasource, viability, longevity and processing, insemination dose media and volume, and insemination timing may be optimized, particularly for low-dose inse'Tunation and potentially high fertility.

Anaddidonal objectofthepresentinvention, according to preferred embodiments, is to provide for the production of amammalthroughhysteroscopic 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 vnth particular embodiments of the invention, blister insemination and bubble or froth irsemination may be introduced as preferred embodiments to optimize fertility rates. A particular goal of the present invention is to provide a technique of Biracial insemination for mammal production utilizing a catheter comprising a videcendoscope for guiding and depositing Me insemination dose.

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

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

Oderobiects ofthe inventionaredisclosedthrougihoutotherareas ofthe specification end claims. In addihon, the goalsandobjecvesmayapply Liberia dependentorindependent fashion to a variety of other goals and objectives in a variety of embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 2 is a diaammatc depiction of the reproductive organs of a female of a mammalian species and, in particular, a depiction of artificial insernmation in accordance win one embodiment of the present invention.

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

MODE() FOR C^RNG ON POSER IRON The basic concepts of Me invention may be embodied in many different ways. The inventive conceptmay involve the materials, elements, apparatus, device andmeods for the production of a mammal through artificial insemination. Furthennore, while one preferred embodiment ofthe invention may be particularly directed to the production of elands through artificial insemination, Me broad concept of Me illYCntiOn should be construed as disclosure of the production of mammals general, and as indicated, to other mamrn1 species such as bovids and swine.

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

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

Accordingly, one embodiment ofthepresentinYention may provideforthecollection of sperm cells from a male of the species of interest. In accordance with one embodiment, sperm cells are collected from one or more stallions of the equine species. According to this embodiment, semen may be collected, and iD prefen ed embodiments, semen ma y be collected with a commercially available artificial vagina, perhaps from at least one stallion of known acceptable fertility. An artificial vagina such as one made available by Animal Reproduction Systems may be used with an in-line gel filter, and inpreferred embodiments, used on alternate days througJloutcollection. ArRercollection, the semen may be evaluated forgel-freevolume, motility, and sperm concentration. In accordance with another preferred embodiment, sperm cells may be collected from othermale species of mammal, particularly that of bovids, equids or swine. An alternative embodiment of the present invention may provide for the collecting of epididymal sperm cells obtained from Me epididyInis of the testis ofthe male species of the maternal. 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, the present invention provides particular embodiments a hysteroscopic insemination sample composing areservoir element, a catheter system to whichthereservoir element isresponsive, arid apluralityofepididymal spermcells contamedwi the reservoir element.

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

Additionally, an artificial insemination sample may be established at volumes, in accordance with preferred embodiments, at volumes between about 30 and I 50 ul, less than about 500 ul, about 230 ul, and about 100 ul. One embodiment ot 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 lS0 ul, less than about 500 ul, about 230 ul, and about 100 al. Furthermore, the present invention is directed to a hysteroscopic insemination sample comprising a reservoir element a catheter system to which the reservoir element is respons*e, and a hysteroscopic compatible volume of sperm contained within said reservoir element. The hysteroscopic compatible volume of sperm contained within said reservoir element may comprise a volume selected from 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 embodiments of the invention, the artificial insemination sample may be placed within a catheter or cathetersystem. In accordance with alternative embodiments, the sample may be placed within a reservoir elemcut or over sample holding element responsive to the catheter or catheter system. A catheter or catheter system should he understood to define any device, system ormethod of insertioninto canals, vessels, passageways, or body cavities to permit injection or withdrawal of fluids, and in accordance with preferred embodnen s such Ejection or withdraw may provide the response of the reservoir element or sample holding element, or lo keep a passage open Preferably, the catheter or catheter system may be used in conjunction with a guide element, and in prefened embodiments an optical element or device, and in preferred embodiments an illumination element, to provide guidance in the artificial nsemirion procedure, as more particularly described below. Preferred embodiments may provide We sobg 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 mL disposable syringe (4) attached to the injection port O on Me distal end (8) of the catheter. The loaded catheter may be withdrawn into a tube, or preferably an outer polypropylene cannula (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. ln accordance with preferred embodiments of the present invention, a determination may be made as to an estrous time for a female McGraw. 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 defined as a state in which the female men mat is capable of conceiving and estrous cycle maybe defined as the correlated phenomena of the er,docrme and generative systems of a female mammal, potentially from the beginning of one period of estrus to not later than the beginning of the next. In accordance with a preferred embodunent, estrus may be induced, and for multiple female mammals synchronized, by administering a substance such as a progestagen, preferably for mares altrenogest, and preferably IOml orally, potentially for 10 consecutive days, followed by 250 tug cloprostenol i.m., potentially on day 11. Furthermore, a female mammal may be induced into ovulation at the time of insemination. Ovulation may be induced, and in preferred embodiments, by the administration of 3000 iu human Chononic Gonadotropin (hCG, Chorulon, Intervet, Inc., Millsboro, Holland), preferably administeredintraveDously at the time of insemination or up to approximately 8 hours previously. Ovulation mar even be induced, in preferred embodiments, by the administration of from about 2000 to about 5000 in human Chononic Gonadotropin.

In preferred embodiments, estrous cycles may be synchronized by administering a synthetic progestagen altrenogest (0.044mg/kg p.o., Regumate; Hoechst Roussel Yet, Warren, New Jersey, USA) daily for 10 consecutive days. Iuteolysis may be induced' in preferred embodiments with the prostaglandin analogue, cloprostenol (250,ugEstmmate, i.m.; Bayer Corporation, Agriculture Division, Shawnee Mission, Kansas, USA) administered on Me eleventh day.

A time 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 in accordance with preferred embodiments a dominant follicle, preferably of> 30mm diameter, is detected. lithe female may be examined until a follicle, preferably pre-ovulatory, of preferably > 35mm is detected. In one embodiment, the female may be inseminated durir g the same day so estrous inducement or synchronization, the same day as ovulation inducement or synchronization, or the same day as estrus and insemination inducement or sychronization.

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

The guide element or optical element (12) and the catheter (2) may then be guided through the vagina (20) of the female, as depicted in Figure 2. In accordance with one embodiment, the optical element (12) and catheter (2) may be manually guided. In fact, the catheter (2) may be manually eluded without the optical element (12). However, accuracy in finding the UTJ and the potential result increased fertility rates, particularly for low numbers of sin and overall potential efficacy m Me procedure, may require a more accurate guidance procedure. Tnpreferred embodiments, the optical element or endoscope, preferably a flexible endoscope, inpreferred embodiments having dimensions of 1.6 m long withan outer diameter of 12 rum, may be guided through the cervix (22) and propelled forward through the uterine lumen (24), orin additional embodiments, through a uterine horn ofthe female ofthe species. An added benefit ofthe use ofthe videoendoscope can be lack of aneed to rectally guide the insemination process, as may have been required in past efforts by those of sicill in the art.

The uterotubal junction (UTJ) (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 the uterotubal junction. In accordance with preferred embodiments, the endoscope may be directed under visual control along the uterine hem (26) ipsilateral to the ovary contamng the pre-ovulatory follicle (28). Preferably the tip ofthe endoscope may be directedproxmate to, and in preferred embodiments within about 3-5cm of said uterotubal junction, and in preferred embodiments within about 3-5 cm of the papilla (32) of the uterotubal junction.

At least a portion of the artificial insemination sample may then be extruded from the catheter (2). In accordance with preferred embodiments, the outer carmuIa (10), followed by the inner GIFT catheter (2) containung the sperm suspension, may be extruded from Me 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 insenunation sample may be aspirated during extrusion Mom the catheter, therebypotendally creating 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 the vicinity of the UTI, but may fi'rther allow for improved fertility rates.

Fermore, an embodiment ofthe present invention provides ahysteroscopic insemination element comprise a uterus of a female species of a mammal, a plurality of sperm cells contained within said uterus, and an aspirated volume of media surrounding or interspersed win the spenn cells and to which the sperm cells are responsive. Such alternative embodiments should be construed as disclosed withregard to all embodiments ofthepresent invention, either in single orin combination, and shouldbe construed to be disclosed as such A low number of sperm may be deposited in die Vichy of the uterotubal junction.

In accordance with preferred embodiments, a plunger of the syringe (4) may be depressed to deposit the sample, and in particular embodiments, a small volume (preferably perhaps 1 00,ll) of the sample, preferably onto Me surface ofthe papilla. The g side element and in preferred embodiments the optical element (12), and the catheter (2) may be withdrawn from the uterus ofthe female mammal. In accordance withpreferred embodiments, filtered air may be introduced within the uterus of the female to facilitate passage ofthe instruments through 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 low number of sperm may be accomplished, in one preferred - embodiment of the invention, by placing with the catheter (2) a number of sperm, preferably numbers selected from: less than about ten nullion sperm, less than about five million spenn, 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 the female may occur and the 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 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% success rates, at least about 50% success rates, at least about 45% success rates, and at least about 90% of a success rate practically experienced with conventional A1 for any particular species. Furthermore, success levels for thepresentinventionmay be statistically comparable to a conventional uterine body artificial insemination process over a sample of cumulative fertilizations whichis greaterdlanabout lOO, whichis greaterthanabout 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.05), therefore potentially being statistically comparable with conventional AI.

Additionally, success rates for the present invention may potentially have the same P value over a range of differing spennnumbers, potentially such as I, 5 or 10 x 106 sperm for equme.

Furthermore, a success level or rate may be expressed ire terms of sample size, whereby the present invention may provide, in preferred embodiments, any ofthe aforementioned success lates over a power calculation At) 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 fertility rates of at least about 90% of those rates resulting from conventional artificial irsenimatio for a species. Additionally, according to particular embodiments of the present invention, the previous success rates mar even be achieved for species of mammal such as bovids, equids, and swine.

In regard to sperm viability, longevity and mobility, for example, thepresent invention may provide for establishing anarcial insemination sample using fresh 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 freezug 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 ire accordance with embodiments of the present invendon herein may permit the use of low numbers of sperm for insermnation even, for example, when using poor quality collected semen.

Alternative embodiments of the present invention may broadly provide for treatment, processing and preserving of insemination sperm. Alternative embodiments of the present invention may provide, for example, for the selection of Me collected sperm cells more likely to achieve insemir atiom According to one embodimentofthe present invention the selection of desired sperm cells or sperm cells more likely to achieve insemination may compris concentrating the more motile sperm collected. Additional embodiments may provide the step of centrifuging the sperm through density gradients, and in preferred embodiments a Percoll gradient. A potentially preferred embodiment may use a 90:45% Percoll gradient. An additional embodiment may comprise limiting the concentration to less Man about twice the starting concentration Implementation of ache Percoll gradient should be understood to encompass die use of Percoll pre orpcstprocessingofthe sperm, and in particular, potentially pre orpost sorting. Therefore, in accordance with embodiments herein, the Percoll gradient procedure may be used with "fresh sperm." lathe 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 Me sample. l he Percoll gradient procedure in accordance win Me present invention may be conducted mconjunctionuaththe use of lower numbers of spermplacedwithinthe depositing catheter, as more particularly described supra Additionally, establishing an artificial msernination sample may be provided by establishing a sample havinghysteroscopic compatible media, thereby providing forpotentially increased fertility rates and an efficacious insemination procedure. More particularly, an embodiment ofthe inventionmayprovide establishing anardficial insemination sample, and in preferred embodiments having a low number sperm compared to natural uasermnation doses, and providing for the establishment of an artificial insemination sample compatible media, for example extender, and in preferred embodiments, a slam milk medium, such as EZ Mixin CST (Animal Reproduction Systems, Chino, CA), preferably as a diluting media, potentially prior to additional processing. In accordance with preferred embodiments of the present invention, dilution may occur to no more Can a 2:1 ratio, to no more than a 5:1 ratio, and to no more than a lO:l ratio, to potentially achieve at least a hysteroscopic compatible volume or media win appropriate concentrations. Furerrnore, embodiments of the present I O invention may provide for the use of an extender, potentially a second extender provided after spermprocessing andpotentiallylnconjunctionwith afirstextender, as previously mentioned, to establish the sample utilizing hysteroscopic compatible media. The second extender may serve to resuspend the sperm sample aPrer processing, and more particularly, provide for a sample utilizing hysteroscopic compatible media. According to preferred embodiments, We medium or second extender may comprise a TALP medium, a HEPES-buffered Tyrode's medium, and an Androhep medium. In accordance with preferred embodiments ofthe present invention, dilution may be performed with a slcim milk medium such as EZ-Mixiu CST@, win a TALP medium, with a HEPES -buffered Tyrode's medium, and with an Androhep medimn, either single or Ln 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 utilizugahysteroscpic compatible media ormediumbaving acatheter 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 OT medium may create an artificial insemination sample having a viscosity of preferably greater Man about that ofthe blood of said mammal or greater than about that of a saline solution. Furthermore additional embodiments may provide for compatible media having a viscosity of greater than about 1 OOcp, a media having a viscosity of greater than about 300cp, a media having a viscosity of greater Han about lOOOcp, a media having a viscosity of greater Man 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 hy$eroscopic insemination sample comprising a reservoir element, a catheter system to which said reservoir element is responsive, a plurality of spend Gel 1 s contained within said reservoi r element; and hysteroscopic compatible media contained within said reservoir element and to which said sperm cells are responsive. In accordance with additional embodimers, 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 the number of sperm cells typically found a natural insemination.

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

One particular embodiment of We 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 prefelTed embodiment of the invention may provide preparation of Me semen through centrifugation. The semen may be diluted to provider for example, 100 x 106 spermatozoa/ml in preferably a commercial skim milk extender (EZ-Mi CST), 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 tune 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 and to potentially select a highly motile fraction of spermatozoa The 90% Percoll may be diluted at a ratio of 1:1 (rev) with HEPES-buffered Tyrode's medium (Grandahl et al., l0 1996) to make a 45% solution. In a preferably 15-mL centrifuge tube, preferably 1 mL of 45% Percoll may be carefully layered on top of preferably I mL of 90% Percoll. Preferably 1 mL ofthe sperm suspension (100 x 1 o6 sperm/mL in EZ-Mixin, CST) may be layered on top of the Percoll layers, and the tube may be centrifuged at 800 x g for aprefened period of 12 minutes. ADcr ccnkifilgation, the supernatant maybe completely removed andthe pelletmay be resuspended in preferably 60Oul HEPES-buffered Tyrode's Medium. The sperm concentration may be deter Tuned, in accordance with one embodiment, usmg a Densimeter (534B MOD-1, Animal Reproduction Systems, Chino, CA) and the potential volume to deliver 5 million spermatozoa (potentially of a preferred 100, l1) may be calculated and prepared for Urination.

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

One embodiment ofthe present inventionmay provide for the collecting of sperm ceils Dom 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 the female is appropriately fertile may be determined, as described above. The optical element ( 12) and the catheter (2) may be inserted vaginally and guided through the vagina, as depicted in Figure 2. The UTJ may be optically located 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. At least a portion of said artificial insemination sample may be extruded under the surface within the vicinity of the UTJ, thus creating a "blister" with 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 Ul T. The surface (34) may comprise the endometrium or other portion of We uterus or the uterine lining. In accordance with one preferred embodiment, the cathetermaybe inserted such that aparticular portion of the uterus is not pierced, and in accordance with one embodiment, such that a vascularized portion of the uterus is not pierced. vascularized portion of the uterus that may not be pierced, according to one particular cnbo&ent, may include, for example, Me mesometrium or the myometrium portion of the uterus or other vascularized portions.

Thereafter, the optical element and catheter may be withdrawn from Me female and 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 ofthepresentinventionmayalsoprovide apotentially corresponding insemination catheter having a guide element, or in preferred embodiments a videcendoscope or a carnula, 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 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 insermnation 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 performed. A low number of sperm may be placed in Me catheter (2), and preferred embodiments, preferably numbers selected from: less than about ten million sperm, less than about five million sperm, less than about two million sperm, less than about one million sperm, less Man about five hundred thousand sperm, and less than about one hundred thousand sperm. Furthermore, the fertilization of an egg may be perfonned accordance with We preferred embodiments of the 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.

Furerrnore, Me sperm cells may be collected Mom a male species of mammal, in alternative embodiments ofthe invention, comprising bovids, equids, or swine. In accordance with alternative embodiments of the invention, sperm cells may be selected Mom collected cells for those cells that may be more likely to achieve insemination, 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, it, accordance with preferred embodiments, a I j nonvascularized portion ofthe uterus, such as The mesometrium or Me myometrium, a cellular cover surface adjacent to the cellular base surface, and in particular embodiments, the endometrium oruterine I ring, a substantial enshrouded volume between Me cellular tease and the cover surface, and in preferred embodiments located the vicinity ofthe UTJ, and sperm cells from the male of the species. Preferred embodiments may also utilize low numbers of sperm relative to natural Lnserrunation, located within the volume and a sperm emission element adjacent the volume through which sperm may pass. lithe 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 comprise a breach in Me endometrium surface of the uterus, as depicted in Figure 3, or may simply occur by diffusion or the like.

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

Such procedures may include sortmg the sperm cells by a sex characteristic, hereby establishing a sex-sorted artificial insemination sample,and inpreferred embodiments having a low number of sperm compared to a natural insemination dosage for said mammal, may include establishing a low dose sex-selected artificial insemination sample. Furthermore, preserving or freezing, and the subsequent Pawing of, sperm sells may be accomplished in particular embodiments, particularly in regard to various mammals such as equid, bovid and swine. Deposition of the msem mation sample, may be processed or aspirated in any way, may be deposited with the crypts or folds of the Ul J. and may provide some type of preservation of the sperm for subsequent insemination. Establishment of an insemination specimen or ulsemination sample at ahysteroscopic compatible volume and utilizing compatible mediamay filcher provide for allowing cooling of the specimen or sample at room temperature.

Cenifugation may preferably be performedthrough 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 be limited to concentrating to less Man about twice the 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 part to the producing of an offspring mammal, may farther be considered to disclose an embodiment of an animal produced utilizing a process as described in any of the foregoing method claims.

Sorting, in accordance with embodiments of the present invention, may particularly provide for collecting sperm cells from amale of a species of mammal, sorting the sperm cells according to a sex-specific characteristic, establishing a sorted, sex-specific artificial insemination sample, placing the sorted, sex-specific artificial insemmabon 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 inseminationtechniques as well as apparatus to accomplish appropriate inseniination. In this application, the 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 natal 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 the foregoing, all of these facets should be

understood to be encompassed by this disclosure.

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

The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many altematiYes are implicit. It also may not Filly explain the generic nature ofthe invention and may not explicitly showhow each feature or element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. Again, these ale implicitly included in this disclosure. Where the invention is described m device-oriented terminology, each element ofthe device implicitly performs a lurction. 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 claims which will be included in a full 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 ofthe various elements ofthe invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood Mat 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 As but one example, it should be understood Mat all actions may be expressed as a means for thing that action or as an element which causes that action. Similarly, each physical element disclosed should be understood to encompass a disclosure of dle action which that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of an 'extruder" should be understood to encompass disclosure of the act of "extruding"-whether explicitly discussed or not -- and, conversely, were there only disclosure of We act of "extruding", such a disclosure should be understood to encompass - disclosure of an "extrudes" and even a "means for extruding". Such changes and alternative terms are to be understood to be explicitly included in the description.

Any acts of law, statutes, regulations, or rules mentionedinthis application for patent, or patents, publications, or other references mentioned in this application for patent, are hereby incorporated by reference. In addition, as to each term used it should be understood that undess its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood as incorporated for each tenn and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictionary, second edition are hereby incorporated by reference. However, as to each of the above, to the extent that such information or statements incorporated by reference might be considered inconsistentwithtle 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 claim at least: i) each of He insemination devices as herein disclosed and described, ii) the related methods disclosed and described, iii) similar,equivalent, and even implicitvariations of eachofese devices and methods, iv) those alternative designs which accomplish each of the Functions shown as are disclosed and described, v) those alternative designs and methods which accomplish each of the functions shown as are implicit to accomplish Mat which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, and ix) methods arid apparatuses substantially as described heremoefore and with reference to any of the accompanying examples, and x) the venous combinations end permutations of each ofthe elements disclosed.

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

It should also be noted that the tend 'fat least one" as used in the following description and claims is not intended nor used in this disclosure to mean that over 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 cLumed, into "closed-ended" clauns wherein devices or methods having additional elements would not be covered by such claims. Accordingly, if or Men used, the use ofthe transitional phrase "comprising" is used to maintain the "open-end" claims herein, according to traditional claim interpretation.

I. PATENT DOCUMENTS DOCUMENT NO. | DATE | NAME/COUNlY | CLASS | SUBCLASS | FILING DATE 5,13 5,759 08/04192 Johnson 424 561 04t26t9 1 6,071,689 06106tOO Sadel d al. 4;5 2 OIJ29t98

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60/238494 OS/IOtOO Morris et aL 05110/00 WO 934094 06108t98 NZ 1. OTHER DOCUMENTS gncludiltg Author, Title, Date, Pertinent Paga, Eta) _. . Bracher, V. and Allen, W.R, 'iVidcoendoscopic Examination of the Mare's Uterus: Fir dings in Normal Fertile Uares", Equine VctaDuy Journal, VoL 24 tl992), pp. 274-278 _. . . . ...

Buchanan, B.R, ct al, "Insemination of Mares with Low Numbers of Either Unsexed or Sexed Spermatozoa", Thedogenology, Vol. 53, W 1333-1344, (ZO00) Cssliclc, B.A., "line Vulva and the Vulvo-vagunal Orifice and its Relation to Genital Health of the Thoroughbred Mare", Cornell Veterinarian, VoL 27, t937, pp. 178-t87 Ctan, D.G., et al, "Production of Lambs by Low Dose Intrauterine Inserninatino with Flow Cytornetrically Sorted and Unsorted Semen", Thedogenology, Vol. 47, pp. 267, (Abstract), (1997) Fugger, E.F., "Clinical Experience with Flow Cytometric Separation of Human X- and YChromosorne Beanng Sperm", Theriogenology, Vol. 52, pp. 1435-1440 (1999) Grondahl, C, et al, 'In Vitro Production of Equine Embryos' Biology of Reproduction, Monograph Series 1, pp. 299307 (1995) - _.. . . Johnson, LA. and Welch, G.R, Sex Preselection: High-speed flow cytometric sorting of X and Y sperm for maximum efficiency", Theriogenology, Vol. 52,(1999),pp. 1323-1341 - - . Johnson, L..A., "Advances in Gender Preselection in Swine" Journal of Reproduction and EertiNty Supplement, Vol. 52, pp. 2SS-266 (1997) Johnson, L..A., "Sex Preselection in Swine: Altered Sex Ratios in Offspring Following Surgical Insenunation of Flow Sorted X-and Y-BeanngSpern' ReproductiDniDDomesDcAnirnals,Vol. 26,pp. 309314 (1991) . . Johnson, L A., et al., "Sex Preselection in Rabbits: Live Birth, from X and Y Spenn Separated by DNA and Cdl Sorting" Biology of Reproduction, VoL 41, pp. 199-203 (1989) Maiming S.T., et al., "Dcvdoprnent otHysteroscopic Insetnination of the Uterine Tucc in the Mare", Proceedings of the Annual Meetmg of the Societv for Theriogenology, 1998, pp 84-85.

Morris, L.H., et al., "Hystercscopic insemination of sawmill numbers of spermatozoa at the uterotubal junction of preovulatory mares", Journal of Reproduction and Fertility, Vo!. 118, pp. 95-100 (2000) Parrish, J.J., et al., "Capacitation of bovine sperm by heparin': Biology of Reproduction, Vol. 38, pp. 1171 -1180 (1988) - . Peippo, I, et al., "Sex diagnosis of equine Reimplantation embryos using the polymerase chain reaction", Theriogccology,Vol. 4461627(1995) 3 0 Pickett GW, et al., "MsDagement of the mare for maximum reproduchve efficiency" Bulletin No. 6 Colorado State University, Ft. Collins CO. (1989) Schenk, J.L., "yopreservatiott of flow-sorted bovme spetmatowa", Iheriogenology, Vol. 52, 1375-1391 (1999) Schrrad ILL., et al, "Fertilization with Sexed Equine Spermatozoa Using Intracytop]asrnic Sperm Injection and Oviductal Insemination I, 7th International Symposium On Equine Reproduction, pp. 139 (Abstract) (1998)

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3 5 Seidel, G.E. Jr, et al., "Artificial Insemination of Heifers with Cooled, Unfrozen Sexed Semen ", Theriogenology Vol. 49 pp. 365 (Abstract) (1998) Seidel, G.E. Jr, et al., insemination of Heifers with Sexed Spenn ", Theriogenology, Vol. 52, pp. 1407-1421 (1999) Squires, }by, '.Early lmblyonic Loss" in Equine Diagnostic Ultrasonography, levied. pp 157163 Bds Rantanen.!k McKinnon. Williams and WiLkins, Banwre, Maryland (1998) Squires, AL.., et al, "Cooled and frozen stallion semen", Bulletin No. 9, Colorado State University, Ft. Collins, CO. (1999) _.

Vazquez, 1., et al., "A.I. in Swine; New Strategy for Deep Insemination with Low Number of Spermatozoa Using a NDn-surgical Methodologyn, 14& Intemational Congress on Animal Reproduction, Vol. 2, Stoclthlorn, July, 2000, p. 289. - .

Vazquez, J., et al., 'onsurgicial Utero4ubal Nomination in Be Mare", Proceedings Annual Meeting of the Society forTheriogenology,1998,pp. 8283.

Vazquez, J., et al., ''Successful Low-Dose Insemination by a Fiberoptic Endoscope Technique in the Sow ' Proceedings Annual Conference of the Intemabonal EimbTyo Transter Society, Netherlands, Theriogenology, Vol. 53, January, 2000, pp 201.

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Vazquez, J., et al., 'evdopment of a Non-surgical Deep Intra Uterine Inserninabon Technique", IV International Conference on Boar Semen Preservation, Maryland, August, 1999, ,o 35 and photo of display board.

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

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

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Johnson, L., et al, "Sex Presclcchon in Swine: Flow Cytometric Sorting of X- and Y- Chromosome Bearing Sperm to Produce Offspring", Boar Semen Preservahon IV, 2000, pp. 107-114.

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Rath, D., et al., "Low Dose Jnseminadon Technique ifl the Pig", Boar Semen Preservation IV, 2000, pp. 115-11B.

Lindsey, A., et al., "Hysteroscopic seminabon of Mares with IVonúrozen Low-dose Unsexed or Sex-sorted Spennatozoa" currently unpublished pp. 115.

Claims (1)

1. Claims for Divisional 4 In the following claims, {be numbenug system from

   the parent case has been left mtact.

   Additions to the clonic (if any) are represented by anmderline. Deletions from the claims (if soy) are represented by a striketbrou' 298. An insemination sample compusing a plurality of sperm cells contained within a reservoir element of a catheter system, wherein sa d sample is cored to be positioned in the vicinity of a uterotubal jnCbQn of a female of a species of a mammal and deposited within the vicinity of the uterotubal junction within said female of said species of said mammal by visually locating the uteotubal junctiam 299. An usenunadon sample as described in claun 298 wherein said sample comprises a hysteroscopic compatible media contained within said reservoir.

   300. An insertion sample as described in claim 298 wherein said sample composes a hysteroscopic compatible volume of sperm contained within said reservoir.

   301. An insemination sample as described in claim 298 wherein said sample con - ses a plurality of frozen thawed sperm cells.

   302. An Insemination sample as described in clam 298 wherein said sample comprises a pluralizer of sorted sperm cells sated according to a sexspecific characteristic.

   303. An usenunaHon sample comprising a plurality of sperm cells, vrherein said sample is configured to be positioned in Me vicmty of a uterobabal junction of a female of a species of a anal and deposited wibin the vicinity of the uterotubal junction within said female of said species of said namrnal by visually locating tiLe uterotubal jncdom 304. An insemination sample as described in claun 303 wherein said sample corpses a bysteroscopic compatible media.

   305. An insemination sample as described claim 303 wherein said sample comprises a hysteroscopic coTnpadble volume of sperm.

   306. An insolation sample as described in claim 303 wherem said sample comprises a plurality of Bow Waived sperm cells.

   307. An insemination sample as described in claim 303 wherein said sample composes a plurality of sorted Sperm cells so according to a sex-specific characteristic. * * *

   309. A method of pmducg a marmnal substantially as hereinbefore described win reference to Me accompanying dravrings.

   310. An insemnatiorr containment element substantially as hereinbefare desenbed with reference deco {be accompanying drann,gs.

   311. An insemination catheter substantially as hereinbefore described with reference to Me accompanyug drawings.

   312. Am mseabon sample substantially as hereinbefore described with reference to the accompanying drawings.

   313. Ahysteroscopio inserninabon sample substantially as hereinbefore described ninth reference to the accompanying drawings.

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

   315. An anunal produced using a process substantially as herembefore described with reference to Me accompanying drawings.