BACKGROUND OF THE INVENTION

Fertilization techniques such as artificial insemination and in vitro fertilization techniques have been conducted in attempt to increase the fertility rates of livestock. Furthermore, effective pre-selection of sex has been accomplished in many species of livestock following the development of safe and reliable methods of sorting, generally, or specifically separating sperm cells into enriched X chromosome bearing and Y chromosome bearing populations. Separation of X chromosome bearing sperm cells from Y chromosome bearing sperm cells, as well as collection, handling, sorting, separation, storage, transportation, use, fertilization, or insemination techniques, or sperm cell and semen processing generally, can be accomplished as disclosed herein and as disclosed by various patent applications, for example: PCT/US99/17165; PCT/US01/45023; PCT/US01/15150; PCT/US98/27909; PCT/US01/45237, PCT/US01/18879, PCT/US/00/30155, PCT/US01/02304, U.S. Pat. No. 6,071,689, U.S. Pat. No. 6,372,422, U.S. divisional application Ser. No. 10/081,955, U.S. provisional application No. 60/400,486, and U.S. provisional application No. 60/400,971, each included in Exhibit A attached, and each hereby incorporated by reference herein.

Although the various devices and methods of sperm cell processing, generally, and the collection, handling, separation, storage, transportation, usage, fertilization, and insemination of sperm cells have been improved over the past several years, significant problems remain with respect to maintaining sperm quality, such as viability, motility, functionality, and preservation and stimulation relative to such techniques, especially with regard to artificial insemination (in vivo) and in vitro fertilization (IVF) procedures. One potential consequence is the reduction in fertility rates. Sperm quality, such as the viability of sperm separated into enriched X-chromosome bearing and Y-chromosome bearing populations could be compared directly in-vitro (for example, in conjunction with IVF procedures) and in-vivo (for example, in conjunction with artificial insemination procedures), is generally reduced during traditional sperm cell processing. More generally, traditional processing techniques addressing sperm viability, motility, functionality, preservation, stimulation, fertilization, and insemination may have not yielded preferred fertility rates, insemination rates, fertilization rates, or sperm quality, generally.

As one example of traditional sperm processing, techniques of sperm sorting for the breeding of mammals may be limited, for example, with regard to sperm cell quality, such as sperm cell viability, and fertility rates, in circumstances wherein the sorter apparatus is a relatively long distance from the males or when the sorter apparatus is a relatively long distance from the receiving females to be inseminated or otherwise fertilized with processed sperm. Some of the techniques incorporated by reference may achieve to some degree sperm cell viability, motility, or functionality, and desirable fertility rates, while addressing, for example, sperm preservation during transportation to the sorter apparatus. However, further techniques achieving sufficient sperm cell quality, such as viability, motility, functionality, stimulation, and preservation, and maintained or enhanced fertility rates, insemination rates, fertilization rates, may be desirable, especially for any one or a combination of various sperm processing steps, such as, for example, collection, handling, separation, storage, transportation, usage, fertilization, or insemination of semen or sperm cells, especially the preservation of the sorted sperm cells from the sorter apparatus to the site of fertilization (potentially via collected individual samples or “straws”), or at any other stage of sperm processing.

As another example of the limitations of traditional sperm processing, traditional processing techniques may be limited with regard to sperm cell quality, generally, such as sperm cell viability, motility, functionality, stimulation, and preservation, as well as fertility rates, insemination rates, fertilization rates, due to the type and the extent processing involved. Known processing techniques such as preservation or sorting, for example, may degrade sperm quality, and further reduce fertility rates, insemination rates, and fertilization rates. The degradation of sperm quality and/or the reduction of fertility rates, insemination rates, and fertilization rates may have previously proven problematic in circumstances that may have required further steps of sperm cell preservation, such as in circumstances wherein the sorter apparatus is a relatively long distance from the males or when the sorter apparatus is a relatively long distance from the receiving females to be inseminated or otherwise fertilized with processed sperm.

It may have been traditionally understood that additional processing steps in the processing of sperm cells or semen having a concentration of sperm cells would degrade sperm quality and reduce fertility rates, insemination rates, and fertilization rates to such an extent that additional processing steps, generally, would be avoided. Specifically, it may also have been traditionally understood that processing steps such as preservation, specifically cryopreservation, and sorting might be too damaging to the sperm cells, especially in processing sequences incorporating both cryopreservation and sorting. Furthermore, it may have even been suggested and taught in traditional methods that preservation of sperm, such as cryopreservation, provided after previous processing steps so as to preserve the sperm for later procedures, such as for in vivo or in vitro techniques, could not be accomplished without compromising the sperm cells and the technique itself to such a degree that the results of the later procedures would be detrimentally affected. Accordingly, such concerns may have even been demonstrated in traditional sperm cell processing procedures, teaching away from subsequent processing steps such as sorting and cryopreservation, or processing steps incorporating multiple cryopreservation steps.

SUMMARY OF THE INVENTION

The present invention addresses the variety of previously identified and potentially unaddressed problems associated with reduced sperm cell quality, such as viability, motility, functionality, stimulation, and of preservation. Sperm cells that may have been or will be processed by one or more steps of collection, handling, separation, storage, transportation, usage, fertilization, or insemination and potential reductions in fertility rates, insemination rates, or fertilization rates are further addressed. The instant invention also addresses the variety of problems associated with sperm cell quality of sperm cells that have been separated into enriched X-chromosome bearing and Y-chromosome bearing populations, potentially sperm cells separated or sorted through techniques such as flow sorting, and the potential reduction in fertility rates. More generally, the present invention addresses traditionally low fertility rates of in vivo artificial insemination and in vitro fertilization, and sperm cell quality issues such as sperm cell viability, motility, functionality, stimulation, and preservation, as well as fertility rates, insemination rates, fertilization rates, achieving results that may have been unexpected to those skilled in the art. Additionally, the present invention addresses numbers of processed sperm potentially needed to obtain desired pregnancy rates, and further in consideration of pregnancy rates obtained respective of sorted and unsorted preserved sperm.

Accordingly, broad objects of the present invention are to provide systems to address sperm cell quality, such as viability, motility, functionality, and preservation, and to address fertilization rates, insemination rates, and fertilization rates. Each of the broad objects of the present invention may be directed to one or more of the various and previously described concerns.

One object of the present invention is to provide methods and systems of semen and sperm cell processing and preservation, and methods and systems of producing a mammal and methods of producing mammalian embryos. A further object of the present invention is to provide preservation, stimulation, fertilization, and insemination, potentially provided alone or in combination with any one or a combination of various sperm processing steps, such as, for example, collection, handling, separation, storage, transportation, usage, fertilization, or insemination of semen or sperm cells. One related object is to provide such systems in relation to the preservation of sperm cells and one or more sequences of sperm cell processing, such as the preservation of sorted sperm cells, and further in some instances, for example, preservation to a sorter apparatus, preservation from a sorter apparatus, preservation to the site of fertilization (potentially via individual sperm samples or “straws”), or at any other stage of sperm processing.

A related object of the invention is to provide systems of collection, handling, separation, storage, transportation, usage, fertilization, or insemination for semen or sperm cells to achieve desirable levels of fertility rates. One related goal is to provide systems of collection, handling, separation, storage, transportation, usage, fertilization, or insemination to maintain and enhance sperm viability, motility, functionality, preservation, stimulation, and levels of fertility rates.

A third object is to address sperm cell quality, such as sperm cell viability, motility, functionality, stimulation, and preservation, as well as fertility rates, in the context of fertilization and insemination, and in some embodiments, in the context of in vivo or in vitro techniques. A related goal of the present invention is to provide systems that achieve desirable levels of fertility rates. A second goal of the present invention is to provide systems that achieve desirable levels of fertility rates in combination with collection, handling, separation, storage, transportation, fertilization, or insemination techniques, and combinations of such techniques. A third goal of the present invention is to provide systems that achieve individual sperm samples of processed sperm cells, such as straws, of desired viability, motility, functionality, preservation, stimulation, or other characteristics, or combinations of characteristics, potentially resulting in desirable levels of fertility rates. Another related goal is to provide systems that achieve sufficient numbers of processed sperm potentially needed to obtain desired pregnancy rates, and further in consideration of pregnancy rates obtained respective of processing techniques, such as the sorting of sperm cells, and in some cases, sperm or sperm cells previously preserved.

A further object is to address sperm cell quality, such as sperm cell viability, motility, functionality, stimulation, and preservation, as well as fertility rates, insemination rates, fertilization rates, for semen or sperm cells obtained from various species of mammals, including, but not limited to equids, bovids, felids, ovids, canids, buffalo, oxen, elk, or porcine; or obtained from prize, endangered, or rare individuals of a mammal species; or obtained from zoological specimens. A related goal of the present invention, therefore, is to address sperm cell quality, generally, such as sperm cell viability, motility, functionality, stimulation, and preservation, as well as fertility rates, insemination rates, fertilization rates, for semen or sperm cells obtained from various species, individuals, and specimens so as to maintain or enhance sperm viability, motility, functionality, preservation, stimulation, fertility rates, or other characteristics, or combinations of characteristics.

Another object of the invention is to provide systems of collection, handling, separation, storage, transportation, usage, fertilization, or insemination for semen or sperm cells obtained from various species of mammals, including, but not limited to equids, bovids, felids, ovids, canids, buffalo, oxen, elk, or porcine; or obtained from prize, endangered, or rare individuals of a mammal species; or obtained from zoological specimens. A related goal of the present invention, therefore, is to provide systems of collection, handling, separation, storage, transportation, usage, fertilization, or insemination for semen or sperm cells obtained from various species, individuals, and specimens so as to maintain or enhance sperm viability, motility, functionality, preservation, stimulation, fertility rates, or other characteristics, or combinations of characteristics.

An object of the present invention is to provide systems that achieve individual samples of processed sperm, such as straws, of desired sperm cell quality, such as sperm cell viability, motility, functionality, stimulation, and preservation, or other characteristics, or combinations of characteristics, as well as providing desirable levels of fertility rates, insemination rates, or fertilization rates.

Another significant object of the invention is to provide systems of sperm cell separation that can maintain or enhance a greater sperm cell quality, such as sperm cell viability, motility, functionality, stimulation, and preservation, or other characteristics, or combination of characteristics, as well as fertility rates, insemination rates, fertilization rates, for sperm cells throughout a flow sorting process such as a process incorporating a flow cytometer.

Another significant object of the invention can be to provide systems of maintaining or enhancing sperm cells at greater sperm cell quality, such as sperm cell viability, motility, functionality, stimulation, and preservation, or other characteristics, or combination of characteristics, as well as fertility rates, insemination rates, or fertilization rates, for purposes of in vivo insemination or in vitro fertilization of various species, such as those described above, or even insemination with a low or reduced number of sperm cells compared to the usual number or typical number of sperm cells used in such insemination procedures whether or not such sperm cells are separated into enriched X chromosome bearing or Y chromosome bearing sperm cells.

Naturally, further significant objects and goals of the invention are disclosed and clarified in the proceeding description of the invention.

Accordingly, to achieve the various objects and goals of the invention, the present invention in one embodiment is a method of sperm cell processing, and the steps provided as obtaining sperm cells, cryopreserving the sperm cells, thawing the sperm cells, processing the sperm cells; and cryopreserving the sorted sperm cells. A further embodiment of the invention is a method of producing a mammal, comprising the method steps of obtaining sperm cells, cryopreserving the sperm cells, thawing the sperm cells, sorting the sperm cells, cryopreserving the sorted sperm cells, thawing the sorted cryopreserved sperm cells, inseminating at least one egg with the sorted cryopreserved sperm cells, fertilizing the at least one egg, and producing a mammal from the at least one fertilized egg.

Furthermore, to achieve the various objects and goals of the invention, the present invention is directed to embodiments providing for artificial insemination (in vivo) and in vitro fertilization procedures. Additionally embodiments may be further directed to the production of mammals or mammalian embryo, and may further be directed to establishing sperm samples such as straws, pellets, or the like, and the processing of semen.

Additional embodiments of the invention are disclosed throughout the description of the invention and in the following claims.

