(12)United States Patent(10)Patent No.: US 8,137,967 B2
 Seidel et al. (45) Date of Patent:Mar.  20, 2012

(54)In-vitro fertilization systems with spermatozoa separated into X-chromosome and Y-chromosome bearing populations 
(75)Inventors: George E. Seidel,  LaPorte, CO (US); 
  Kehuan Lu,  Nanning (CN); 
  Tae Kwang Suh,  Fort Collins, CO (US) 
(73)Assignee:XY, LLC,  Navasota, TX (US), Type: US Company 
(*)Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 840 days. 
(21)Appl. No.: 11/508,133 
(22)Filed: Aug.  21, 2006 
(65)Prior Publication Data 
 US 2006/0281176 A1 Dec.  14, 2006 
 Related U.S. Patent Documents 
(63) .
Continuation of application No. 10/433,191, now Pat. No. 7,094,527 .
(60)Provisional application No. 60/253,787, filed on Nov.  29, 2000.
 Provisional application No. 60/253,785, filed on Nov.  29, 2000.
(51)Int. Cl. C12N 005/02 (20060101)
(52)U.S. Cl. 435/373; 435/363; 435/325; 435/2
(58)Field of Search  435/2, 325, 373, 363

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 5,880,457  A  3/1999    Tomiyama et al.     
 5,880,474  A  3/1999    Norton et al.     
 5,883,378  A  3/1999    Irish et al.     
 5,888,730  A  3/1999    Gray et al.     
 5,891,734  A  4/1999    Gill et al.     
 5,893,843  A  4/1999    Rodrigues Claro     
 5,895,764  A  4/1999    Sklar et al.     
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 5,899,848  A  5/1999    Haubrich     
 5,909,278  A  6/1999    Deka et al.     
 5,912,257  A  6/1999    Prasad et al.     
 5,916,144  A  6/1999    Prather et al.     
 5,916,449  A  6/1999    Ellwart et al.     
 5,917,733  A  6/1999    Bangham     
 5,919,360  A  7/1999    Contaxis, III et al.     
 5,919,621  A  7/1999    Brown     
 5,934,885  A  8/1999    Farrell et al.     
 5,962,238  A  10/1999    Sizto et al.     
 5,972,710  A  10/1999    Weigl et al.     
 5,973,842  A  10/1999    Spangenberg     
 5,985,216  A  11/1999    Rens et al.     
 5,985,538  A  11/1999    Stachecju     
 5,990,479  A  11/1999    Weiss et al.     
 5,991,028  A  11/1999    Cabib et al.     
 5,998,140  A  12/1999    Dervan et al.     
 5,998,212  A  12/1999    Corio et al.     
 6,002,471  A  12/1999    Quake     
 6,003,678  A  12/1999    Van den Engh     
 6,042,025  A  3/2000    Crampton et al.     
 6,042,249  A  3/2000    Spangenberg     
 6,050,935  A  4/2000    Ranoux et al.     
 6,071,689  A  6/2000    Seidel et al.     
 6,079,836  A  6/2000    Burr et al.     
 6,086,574  A  7/2000    Carroll et al.     
 6,087,352  A  7/2000    Trout     
 6,090,947  A  7/2000    Dervan et al.     
 6,097,485  A  8/2000    Lievan     
 6,111,398  A  8/2000    Graham     
 6,117,068  A  9/2000    Gourley et al.     
 6,119,465  A  9/2000    Mullens et al.     
 6,120,735  A  9/2000    Zborowski et al.     
 6,128,133  A  10/2000    Bergmann     
 6,130,034  A  10/2000    Aitken     
 6,132,961  A  10/2000    Gray et al.     
 6,133,044  A  10/2000    Van den Engh     
 6,133,995  A  10/2000    Kubota     
 6,139,800  A  10/2000    Chandler     
 6,140,121  A  10/2000    Ellington et al.     
 6,143,535  A  11/2000    Paisson     
 6,143,901  A  11/2000    Dervan     
 6,146,837  A  11/2000    van de Winkel     
 6,149,867  A  11/2000    Seidel et al.     
 6,153,373  A*11/2000    Benjamin et al. 435/2
 6,154,276  A  11/2000    Mariella, Jr.     
 6,175,409  B1  1/2001    Nielsen et al.     
 6,177,277  B1  1/2001    Soini     
 6,193,647  B1  2/2001    Beebe et al.     
 6,201,628  B1  3/2001    Basiji et al.     
 6,207,392  B1  3/2001    Weiss et al.     
 6,208,411  B1  3/2001    Vaez-Iravani     
 6,211,477  B1  4/2001    Cardott et al.     
 6,214,560  B1  4/2001    Yguerabide et al.     
 6,221,654  B1  4/2001    Quake et al.     
 6,221,671  B1  4/2001    Groner et al.     
 6,238,920  B1  5/2001    Nagai et al.     
 6,247,323  B1  6/2001    Maeda     
 6,248,590  B1  6/2001    Malachowski     
 6,256,096  B1  7/2001    Johnson     
 6,263,745  B1  7/2001    Buchanan et al.     
 6,283,920  B1  9/2001    Eberle et al.     
 6,296,810  B1  10/2001    Ulmer     
 6,309,815  B1  10/2001    Tash et al.     
 6,316,234  B1  11/2001    Bova     
 6,317,511  B1  11/2001    Horiuchi     
 6,322,901  B1  11/2001    Bawendi et al.     
 6,323,632  B1  11/2001    Husher et al.     
 6,326,144  B1  12/2001    Bawendi et al.     
 6,328,071  B1  12/2001    Austin     
 6,329,158  B1  12/2001    Hoffman et al.     
 6,332,540  B1  12/2001    Paul et al.     
 6,357,307  B2  3/2002    Buchanan et al.     
 6,368,786  B1  4/2002    Saint-Ramon et al.     
 6,372,422  B1  4/2002    Seidel et al.     
 6,372,506  B1  4/2002    Norton     
 6,384,951  B1  5/2002    Basiji et al.     
 6,395,305  B1  5/2002    Buhr et al.     
 6,400,453  B1  6/2002    Hansen     
 6,411,835  B1  6/2002    Modell et al.     
 6,411,904  B1  6/2002    Chandler     
 6,416,190  B1  7/2002    Grier et al.     
 6,423,505  B1  7/2002    Davis     
 6,423,551  B1  7/2002    Weiss et al.     
 6,432,630  B1  8/2002    Blankenstein     
 6,432,638  B2  8/2002    Dervan et al.     
 6,452,372  B1  9/2002    Husher et al.     
 6,454,945  B1  9/2002    Weigl et al.     
 6,456,055  B2  9/2002    Shinabe et al.     
 6,463,314  B1  10/2002    Haruna     
 6,465,169  B2  10/2002    Walderich et al.     
 6,473,176  B2  10/2002    Basiji et al.     
 6,482,652  B2  11/2002    Furlong et al.     
 6,489,092  B1  12/2002    Benjamin et al.     
 6,495,333  B1  12/2002    Willmann et al.     
 6,495,366  B1  12/2002    Briggs     
 6,503,698  B1  1/2003    Dobrinsky et al.     
 6,511,853  B1  1/2003    Kopf-Sill et al.     
 6,514,722  B2  2/2003    Paisson et al.     
 6,524,860  B1  2/2003    Seidel et al.     
 6,528,802  B1  3/2003    Koenig et al.     
 6,534,308  B1  3/2003    Palsson et al.     
 6,537,829  B1  3/2003    Zarling et al.     
 6,540,895  B1  4/2003    Spence et al.     
 6,563,583  B2  5/2003    Ortyn et al.     
 6,576,291  B2  6/2003    Bawendi et al.     
 6,577,387  B2  6/2003    Ross, III et al.     
 6,580,504  B1  6/2003    Ortyn et al.     
 6,587,203  B2  7/2003    Colon     
 6,589,792  B1  7/2003    Malachowski     
 6,590,911  B1  7/2003    Spinelli et al.     
 6,596,143  B1  7/2003    Wang et al.     
 6,596,499  B2  7/2003    Jalink     
 6,604,435  B2  8/2003    Buchanan et al.     
 6,613,525  B2  9/2003    Nelson et al.     
 6,617,107  B1  9/2003    Dean     
 6,618,143  B2  9/2003    Roche et al.     
 6,618,679  B2  9/2003    Loehrlein et al.     
 6,641,708  B1  11/2003    Becker et al.     
 6,642,018  B1  11/2003    Koller et al.     
 6,658,357  B2  12/2003    Chandler     
 6,664,550  B2  12/2003    Rader et al.     
 6,667,830  B1  12/2003    Iketaki et al.     
 6,671,044  B2  12/2003    Ortyn et al.     
 6,673,095  B2  1/2004    Nordquist     
 6,674,525  B2  1/2004    Bardell et al.     
 6,698,627  B2  3/2004    Garcia et al.     
 6,700,130  B2  3/2004    Fritz     
 6,703,621  B2  3/2004    Wolleschensky     
 6,704,313  B1  3/2004    De Resende et al.     
 6,706,163  B2  3/2004    Seul et al.     
 6,707,555  B1  3/2004    Kusuzawa et al.     
 6,713,019  B2  3/2004    Ozasa et al.     
 6,729,369  B2  5/2004    Neas et al.     
 6,746,873  B1  6/2004    Buchanan et al.     
 6,752,298  B2  6/2004    Garcia et al.     
 6,753,161  B2  6/2004    Koller et al.     
 6,761,286  B2  7/2004    Py et al.     
 6,761,288  B2  7/2004    Garcia     
 6,767,706  B2  7/2004    Quake     
 6,780,377  B2  8/2004    Hall et al.     
 6,782,768  B2  8/2004    Buchanan et al.     
 6,789,706  B2  9/2004    Abergel et al.     
 6,789,750  B1  9/2004    Heldt     
 6,793,387  B1  9/2004    Neas et al.     
 6,813,017  B1  11/2004    Hoffman et al.     
 6,819,411  B1  11/2004    Sharpe et al.     
 6,849,394  B2  2/2005    Rozenboom et al.     
 6,849,423  B2  2/2005    Mutz et al.     
 6,861,265  B1  3/2005    Van den Engh     
 6,941,005  B2  9/2005    Lary et al.     
 7,015,310  B2  3/2006    Remington et al.     
 7,105,355  B2  9/2006    Kurabayashi et al.     
 7,208,265  B1  4/2007    Schenk     
 7,221,453  B2  5/2007    Sharpe et al.     
 2001//0006416  A1  7/2001    Johnson     
 2002//0047697  A1  4/2002    Husher et al.     
 2002//0058332  A1  5/2002    Quake et al.     
 2002//0064809  A1  5/2002    Mutz et al.     
 2002//0096123  A1  7/2002    Whittier et al.     
 2002//0113965  A1  8/2002    Roche et al.     
 2002//0115055  A1  8/2002    Matta     
 2002//0119558  A1  8/2002    Seidel et al.     
 2002//0131957  A1  9/2002    Gavin     
 2002//0141902  A1  10/2002    Ozasa et al.     
 2002//0171827  A1  11/2002    Van den Engh     
 2002//0182590  A1  12/2002    Strange et al.     
 2002//0186375  A1  12/2002    Asbury et al.     
 2002//0186874  A1  12/2002    Price et al.     
 2002//0198928  A1  12/2002    Bukshpan et al.     
 2003//0002027  A1  1/2003    Fritz     
 2003//0048433  A1  3/2003    Desjonqueres     
 2003//0059764  A1  3/2003    Ravkin et al.     
 2003//0059945  A1  3/2003    Dzekunov et al.     
 2003//0078703  A1  4/2003    Potts et al.     
 2003//0096405  A1  5/2003    Takayama et al.     
 2003//0098421  A1  5/2003    Ho     
 2003//0113765  A1  6/2003    Dempcy et al.     
 2003//0119050  A1  6/2003    Shai     
 2003//0119206  A1  6/2003    Shai     
 2003//0129091  A1  7/2003    Seidel et al.     
 2003//0157475  A1  8/2003    Schenk     
 2003//0165812  A1  9/2003    Takayama et al.     
 2003//0175917  A1  9/2003    Cumming     
 2003//0175980  A1  9/2003    Hayenga et al.     
 2003//0190681  A1  10/2003    Shai     
 2003//0207461  A1  11/2003    Bell et al.     
 2003//0209059  A1  11/2003    Kawano     
 2004//0005582  A1  1/2004    Shipwast     
 2004//0031071  A1  2/2004    Morris et al.     
 2004//0034879  A1  2/2004    Rothstein et al.     
 2004//0049801  A1  3/2004    Seidel     
 2004//0053243  A1  3/2004    Evans     
 2004//0055030  A1  3/2004    Maxwell et al.     
 2004//0061070  A1  4/2004    Hansen     
 2004//0061853  A1  4/2004    Blasenheim     
 2004//0062685  A1  4/2004    Norton et al.     
 2004//0072278  A1  4/2004    Chou et al.     
 2004//0107150  A1  6/2004    Neas et al.     
 2004//0132001  A1  7/2004    Seidel et al.     
 2005//0003472  A1  1/2005    Anzar et al.     
 2005//0011582  A1  1/2005    Haug     
 2005//0064383  A1  3/2005    Bashkin et al.     
 2005//0112541  A1  5/2005    Durack     
 2005//0214733  A1  9/2005    Graham     
 2005//0244805  A1  11/2005    Ludwig et al.     
 2005//0282245  A1  12/2005    Ludwig et al.     
 2006//0118167  A1  6/2006    Neas et al.     
 2006//0147894  A1  7/2006    Sowter     
 2006//0203226  A1  9/2006    Roche et al.     
 2006//0263829  A1  11/2006    Evans et al.     
 2006//0281176  A1  12/2006    Seidel et al.     
 2007//0026378  A1  2/2007    Schenk     
 2007//0026379  A1  2/2007    Seidel et al.     
 2007//0042342  A1  2/2007    Seidel et al.     
 2007//0092860  A1  4/2007    Schenk     
 2007//0099171  A1  5/2007    Schenk     
 2007//0099260  A1  5/2007    Seidel et al.     
 2007//0117086  A1  5/2007    Evans et al.     
 2007//0123461  A1  5/2007    Josephson     
 2007//0248976  A1  10/2007    Harding     

       BR       9704313                         6/1999      
       CA       1029833                         4/1978      
       CA       1 250 808                         3/1989      
       CA       2113957       A1                1/1994      
       CN       100998524                         7/2007      
       DE       69028526                         2/1997      
       DE       195 49 015       C1                4/1997      
       DE       198 82 943.3                         2/2001      
       EP       0025296       A2                3/1981      
       EP       0 046 345       A2                2/1982      
       EP       0 068 404       B1                1/1983      
       EP       0071538       A1                2/1983      
       EP       0 026 770       B1                3/1983      
       EP       0 029 662       B1                2/1984      
       EP       0 025 296       B1                5/1985      
       EP       0140616                         5/1985      
       EP       0 158 147       A2                10/1985      
       EP       0160201       A2                11/1985      
       EP       0189702       A1                8/1986      
       EP       0 229 814       B1                7/1987      
       EP       0 246 604       A2                11/1987      
       EP       0288029       B1                4/1988      
       EP       0276166       A2                7/1988      
       EP       0 289 677       A2                11/1988      
       EP       0 316 173       A1                5/1989      
       EP       0 317 809       A2                5/1989      
       EP       A-0 366794                         5/1990      
       EP       0 409 293       A2                1/1991      
       EP       0461618                         12/1991      
       EP       0 463 562       A1                1/1992      
       EP       0468100       A1                1/1992      
       EP       0474 187       A2                3/1992      
       EP       0 316 172       B1                7/1992      
       EP       0 316 171       B1                9/1992      
       EP       0570102       A1                3/1993      
       EP       0538786       A                4/1993      
       EP       0 279 000       B1                7/1993      
       EP       0 553 951       A1                8/1993      
       EP       0 288 029       B1                1/1994      
       EP       0 381 694       B1                6/1994      
       EP       0 361 504       B1                7/1994      
       EP       606847       A2                7/1994      
       EP       0 289 200       B2                8/1994      
       EP       0 555 212       B1                10/1994      
       EP       0 361 503       B1                11/1994      
       EP       0 696 731       A2                2/1996      
       EP       0 705 978       A2                4/1996      
       EP       0 711 991       A1                5/1996      
       EP       0 471 758       B1                9/1996      
       EP       0 736 765       A1                10/1996      
       EP       0 545 284       B1                2/1997      
       EP       0 360 487       B1                7/1997      
       EP       0 412 431       B1                10/1997      
       EP       0 526 131       B1                1/1998      
       EP       A-0 478155                         1/1998      
       EP       0 822 404       A3                2/1998      
       EP       0 822 401       A2                4/1998      
       EP       0781985       A3                7/1998      
       EP       0 556 748       B1                10/1998      
       EP       0 430 402       B1                1/1999      
       EP       0 529 666       B1                4/2000      
       EP       0 994 342       A3                4/2000      
       EP       0 752 133       B1                6/2000      
       EP       1 018 644       A2                7/2000      
       EP       1 118 268       A1                7/2001      
       EP       1 147 774       A1                10/2001      
       EP       0 534 033       B1                11/2001      
       EP       0 925 494       B1                12/2001      
       EP       0 748 316       B1                5/2002      
       EP       0 662 124       B1                6/2002      
       EP       1 245 944       A3                10/2002      
       EP       1 249 502       A2                10/2002      
       EP       1250897       A1                10/2002      
       EP       1 380 304       A2                1/2004      
       EP       1403633       A3                4/2004      
       EP       1 100 400       B1                5/2004      
       EP       1 257 168       B1                2/2005      
       FR       2574656       A1                6/1986      
       FR       A-2 635453                         2/1990      
       FR       2 647 668       A                12/1990      
       FR       2699678       A1                6/1994      
       GB       1471019       A1                4/1977      
       GB       2 121 976       A                1/1984      
       GB       2 122 369       A                1/1984      
       GB       2 125 181       A                2/1984      
       GB       2 136 561       A                9/1984      
       GB       2 137 352       A                10/1984      
       GB       2145112                         2/1985      
       GB       2 144 542       A                3/1985      
       GB       2 153 521       A                8/1985      
       GB       2 243 681       A                11/1991      
       GB       2 360 360       A                9/2001      
       JP       61139747       A                6/1986      
       JP       61159135       A                7/1986      
       JP       2024535                         1/1990      
       JP       4126064       A                4/1992      
       JP       4126065       A                4/1992      
       JP       4126066       A                4/1992      
       JP       4126079       A                4/1992      
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  Habermann F. A., et al., Validation of sperm sexing in the cattle (Bos taurus) by dual colour flourescence in situ hybridization; J Anim Breed Genet. Apr. 2005; 122 Suppl 1:22-7 (Abstract only).
  Johnson, L. A., Sexing mammalian sperm for production of offspring: the state-of-the-art; Animal Reproduction Science; 60-61 (2000) pp. 93-107.
  Parallel Canada Patent Application No. 2468774; Office action dated Aug. 11, 2009.
  Parallel Chilean application No. 3016-01, Office Action dated Jan. 6, 2010, 4 pages.
  Abeydeera et al. Birth of Piglets Preselected for Gender Following in Vitro Fertilization on in Vitro Matured Pig Oocytes by X and Y Chromosome Dearing Spermatozoa Sorted by High Speed Flow Cytometry. Theriogenology 50: 981-988, 1998.
     * cited by examiner
     Primary Examiner —Ruth Davis
     Art Unit — 1651
     Exemplary claim number — 1
(74)Attorney, Agent, or Firm — Craig R. Miles; CR Miles, P.C.



An IVF system for successfully utilizing spermatozoa separated into X-chromosome bearing and into Y-chromosome bearing population for insemination. The IVF system includes fertilization medium that can shorten the time from insemination to cleavage and a portable incubator for the transportation of maturing oocytes and inseminated oocytes comprising a straw (19) and an incubation element (20) that can be sealed with a cap (22).
12 Claims, 4 Drawing Sheets, and 4 Figures

[0001] This application is a continuation of application Ser. No. 10/433,191, filed May 29, 2003, issuing Aug. 22, 2006 as U.S. Pat. No. 7,094,527, which is the National Stage of International Application No. PCT/US01/45237, filed Nov. 29, 2001, which claims the benefit of under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/253,787, filed Nov. 29, 2000 and U.S. Provisional Patent Application No. 60/253,785, filed Nov. 29, 2000, each hereby incorporated by reference herein.


[0002] Devices, compositions, and methods that improve the quality of embryos generated using in-vitro fertilization (IVF) with spermatozoa separated into X-chromosome bearing and Y-chromosome bearing populations.


[0003] An attractive feature of IVF is that many fewer spermatozoa can be required for insemination than for artificial insemination. However, IVF using spermatozoa separated into X-chromosome bearing and Y-chromosome bearing populations (separated spermatozoa) can necessitate modifications to conventional IVF techniques. This may be due in part to the pre-capacitation of such spermatozoa.
[0004] In most cases, the percentages of oocytes (oocyte, ootid, or ova, or plurality of same as appropriate to the application) fertilized with separated and unseparated spermatozoa are similar, and events during the first cell cycle are timed similarly for separated and unseparated spermatozoa. However, with conventional procedures, blastocyst production with separated spermatozoa can be 70%-90% of controls with spermatozoa that have not been separated. For example, development to blastocysts has been shown to be 17% with bovine oocytes inseminated with separated spermatozoa, compared with >25% which might be expected with IVF using unseparated spermatozoa as described in the journal article entitled “In Vitro Fertilization With Flow-Cytometerically-Sorted Bovine Sperm” Theriogenology 52: 1393-1405 (1999), hereby incorporated by reference.
[0005] Several factors may contribute to these results. One factor may be that staining of sperm with Hoechst 33342 appears to cause a decline in motility of spermatozoa. Another factor, may be the physical forces the spermatozoa are subject to during the separation process. As but one example, in flow cytometric separation of spermatozoa, spermatozoa exit the flow cytometer at nearly 100 km/h before impacting on the surface of the collection medium. During transit through the flow cytometer spermatozoa can be subjected to laser light at an intensity of over 100 mW. While the transit time may only be 1-2 μsec, this may affect the spermatozoal DNA, and thus, also effect subsequent embryonic development. The process of separating sperm with flow cytometry can also result in a highly diluted sample, 600,000 spermatozoa/mL or less, and subsequent centrifugation steps are necessary to provide concentrated spermatozoa suitable for insemination.
[0006] Another problem with utilizing separated spermatozoa in IVF techniques may be that the facility in which the spermatozoa are separated may be in a different location than where the male mammal from which the spermatozoa are collected is located, which may be different from where the female mammal from which the oocytes are collected is located, which may be a different location from where the in-vitro fertilization is to occur, and which may be a different location from where the female mammal into which the in-vitro cultured embryos are to be transferred. Conventionally, separated sperm may be cryopreserved and transported frozen to the facility at which the IVF techniques are administered. Maturing oocytes are conventionally transported to the facility at which the IVF techniques are administered in portable incubation systems. The maturing oocytes are then inseminated with previously frozen-thawed sperm cells. To avoid cryopreservation of sperm cells or as a convenience to the various facilities involved it may be beneficial to transport maturing oocytes directly to the facility separating the spermatozoa so that separated sperm cells can be added to the oocytes without cryopreservation. However, conventional IVF and in vitro culture of the resulting zygotes typically comprises a separate set of apparatus and procedures making it inconvenient, difficult, or impossible to inseminate and culture oocytes in the same facility in which spermatozoa are separated.
[0007] Even though X-chromosome bearing spermatozoa and Y-chromosome bearing spermatozoa have been differentiated by and separated based upon the difference in emitted fluorescence for many years, and even though separated spermatozoa have been used for some time with IVF techniques, and even though there is large commercial market for embryos produced with IVF techniques and separated spermatozoa, the above-mentioned problems have yet to be resolved.
[0008] As to the problems with conventional techniques of IVF using separated spermatozoa, and specifically separated spermatozoa, stained spermatozoa, or spermatozoa that are from previously frozen sperm, and with conventional strategies involving the transportation of separated sperm and maturing oocytes, the invention addresses each in a practical manner.


[0009] Accordingly, one of the broad objects of particular embodiments of the invention can be to provide devices, compositions and methods that provide transportation of inseminated oocytes, promotes cleavage of fertilized oocytes and improves the quality of embryos generated with techniques utilizing spermatozoa separated into X-chromosome bearing and Y-chromosome bearing populations.
[0010] Another broad object of particular embodiments of the invention can be to provide devices, compositions, and methods that promote cleavage and improve quality of embryos generated using IVF with spermatozoa that are derived from previously frozen sperm.
[0011] Another broad object of particular embodiments of the invention can be to provide devices, compositions, and methods that promote cleavage and improve quality of embryos generated using IVF with spermatozoa that have previously been stained with a DNA binding fluorochrome.
[0012] Another broad object of particular embodiments of the invention can be to provide medium for embryonic culturing that can contain non-essential amino acids.
[0013] Another broad object of the invention can be to provide apparatus and methods for transporting maturing oocytes and fertilized oocytes for the convenience of the end user(s) or to avoid cryopreservation of the spermatozoa used to fertilize oocytes.
[0014] Naturally further objects of the invention are disclosed throughout other areas of specification.


[0015] FIG. 1 shows an embodiment of the invention in which spermatozoa from fresh or previously frozen-thawed sperm are stained.
[0016] FIG. 2 shows an embodiment of the invention for separating stained spermatozoa in to X-chromosome bearing and Y-chromosome bearing populations.
[0017] FIG. 3 shows another view of an embodiment of the invention for separating stained spermatozoa in to X-chromosome bearing and Y-chromosome bearing populations.
[0018] FIG. 4 shows an embodiment of a portable incubation system in which oocytes can be fertilized.


[0019] The invention involves devices, methods, and compositions for the in-vitro insemination and fertilization of oocytes (oocyte, ootid, or ova, or plurality of same as appropriate to the application) and the culture of embryos resulting from such techniques.
[0020] Embodiments of the invention can include fresh spermatozoa, or spermatozoa from frozen-thawed sperm of numerous species of mammals. The invention should be understood not to be limited to the species of mammals cited by the specific examples within this patent application. Embodiments of the invention, for example, may include fresh spermatozoa or spermatozoa from frozen-thawed sperm of animals having commercial value for meat or dairy production such as swine, bovids, ovids, equids, buffalo, or the like (naturally the mammals used for meat or dairy production may vary from culture to culture). It may also include fresh spermatozoa or spermatozoa from frozen-thawed sperm from individuals having rare or uncommon attribute(s), such as morphological characteristics including weight, size, or conformation, or other desired characteristics such as speed, agility, intellect, or the like. It may include frozen-thawed sperm from deceased donors, or fresh or frozen-thawed spermatozoa from rare or exotic mammals, such as zoological specimens or endangered species. Embodiments of the invention may also include fresh or frozen-thawed spermatozoa collected from primates, including but not limited to, chimpanzees, gorillas, or the like, and may also include fresh or frozen-thawed spermatozoa from marine mammals, such as whales or porpoises.
[0021] Now referring primarily to FIG. 1, in some embodiments of the invention, Hoechst 33342 stain (1) can be added to bovine spermatozoa contained in frozen-thawed sperm (2) to establish a concentration of 224 μM. The incubation time of the spermatozoa contained in the frozen-thawed sperm (2) with the stain (1) can be about 190 minutes. In anther embodiment of the invention, the stain (1) can be added to the bovine sperm (2) to establish a concentration of 2240 μM and then incubated for about 60 minutes. Frozen-thawed sperm treated in either manner can improve the resolution of X-chromosome bearing from Y-chromosome bearing spermatozoa. Understandably, from application to application (such as frozen-thawed sperm from different species) the amount of incubation time and the specific concentration of stain can be adjusted to optimize the resolution of the X-chromosome bearing from Y-chromosome bearing spermatozoa.
[0022] With respect to the cleavage rates of inseminated oocyte(s), the increase in stain concentration up to at least 10× does not appear to have a depressive effect on either cleavage or embryonic development. Higher stain concentrations may actually be beneficial with respect to certain applications because the length of incubation time may be decreased improving percent cleavage. From application to application length of incubation time can be adjusted to optimize cleavage results or embryonic development, as desired.
[0023] Now referring primarily to FIGS. 2 and 3, a flow cytometer embodiment of the invention is shown which includes a sperm cell source (3) which acts to establish or supply stained spermatozoa or other type of stained cells to be analyzed by the flow cytometer. The sperm cells are deposited within a nozzle (4) in a manner such that the cells are surrounded by a sheath fluid (5). The sheath fluid (5) is usually supplied by some sheath fluid source (6) so that as the cell source (3) supplies sperm cells, the sheath fluid (5) is concurrently fed through the nozzle (4). In this manner it can be easily understood how the sheath fluid (5) forms a sheath fluid environment for the cells. Since the various fluids are provided to the flow cytometer at some pressure, they flow out of the nozzle (4) and exit at the nozzle orifice (7). By providing some type of oscillator (8) which may be very precisely controlled through an oscillator control (9), pressure waves may be established within the nozzle (4) and transmitted to the fluids exiting the nozzle (4) at nozzle orifice (7). Since the oscillator (9) thus acts upon the sheath fluid (5), the stream (10) exiting the nozzle orifice (7) eventually and regularly forms drops (11). Because the sperm cells are surrounded by a sheath fluid environment, the drops (11) may contain within them individually isolated (generally) cells or other items.
[0024] Since the drops (11) generally contain isolated sperm cells, the flow cytometer can distinguish and separate droplets based upon whether or not the appropriate sperm cell is contained within the drop. This is accomplished through a cell sensing system (12). The cell sensing system involves at least some type of sensor (14) which responds to the cells contained within each drop (11) as described by U.S. Pat. No. 5,135,759, hereby incorporated by reference. As the Johnson patent explains for spermatozoa or sperm cells, although the staining and separation inventions can be understood to be used with a variety of frozen-thawed cells, the cell sensing system (12) may cause an action depending upon the relative presence or relative absence of the bound fluorochrome which may be excited by some stimulant such as the laser exciter (13). While each type of sperm cell can be stained by the stain or fluorochrome, as described above, the differing length of the X-chromosome and the Y-chromosome causes different amounts of stain to be bound. Thus, by sensing the degree of fluorescence emitted by the fluorochrome upon excitation it is possible to discriminate between X-bearing spermatozoa and Y-bearing spermatozoa by their differing fluoresence emission levels.
[0025] In order to achieve separation and isolation of the appropriate sperm cells, the signals received by sensor (14) are fed to some type of sorter discrimination system (15) which very rapidly makes a differentiation decision and can differentially charge each drop (11) based upon whether it has decided that the desired sperm cell does or does not exist within that drop (11). In this manner the separation or discrimination system (15) acts to permit the electrostatic deflection plates (16) to deflect drops (11) based on whether or not they contain the appropriate sperm cell. As a result, the flow cytometer acts to sort the sperm cells by causing them to land in one or more collectors (17). Thus by sensing some property of the sperm cells the flow cytometer can discriminate between sperm cells based on a particular characteristic and place them in the appropriate collector (17). In the system presently used to sort spermatozoa, the X-bearing spermatozoa droplets are charged positively and thus deflect in one direction, the Y-bearing spermatozoa droplets are charged negatively and thus deflect the other way, and the wasted stream (that is unsortable cells) is uncharged and thus is collected in an undeflected stream into a suction tube or the like.
[0026] Now referring primarily to FIG. 3, the process can be even further understood. As shown in that figure, the nozzle (4) emits a stream (10) which because of the oscillator (8) (not shown in FIG. 3) forms drops (11). Since the cell source (3) (not shown in FIG. 3) may supply sperm cells (1) which have been stained according the invention, the magnitude of the fluorescent emission stimulated by the laser exciter (13) is differentially determined by sensor (14) so that the existence or nonexistence of a charge on each drop (11) as it separates from stream (10) can be controlled by the flow cytometer. This control results in positively charged, negatively charged, and uncharged drops based upon the encapsulated sperm cell. As shown in FIG. 3, certain drops are shown as deflected drops (18). These deflected drops (18) are those containing sperm cells (2) differentiated by bearing an X-chromosome or a Y-chromosome. Separated sperm are then deposited in the appropriate collector (17) for later use. See also, International Patent Application PCT/US98/27909, hereby incorporated by reference.
[0027] While the above description focuses on the separation of spermatozoa with flow cytometry, separation of X-chromosome bearing spermatozoa and Y-chromosome bearing spermatozoa based upon the difference in measurable fluorescent emission may also include numerous other technologies such as liquid chromatography, gel electrophoresis, and other technologies that similarly excite the amount of bound fluorochrome to differentiate between X chromosome bearing spermatozoa and the Y chromosome bearing spermatozoa.
[0028] Embodiments of the invention can also comprise collecting oocytes from a female mammal. With respect to certain embodiments of the invention, oocytes can be aspirated from the ovaries of the desired female mammal or can be obtained from slaughterhouse ovaries. The oocytes can be matured in TCM199 supplemented with about 10% fetal calf serum plus hormones (15 ng FSH, 1 μg LH, 1 μg E2/ml) for 22-24 h at 39° C., in about 5% CO2 in air.
[0029] Ten to 15 oocytes can be transferred to a 50 μl drop of fertilization medium containing non-essential amino acids, such as tyrode albumin lactaate pyruvate (TALP) supplemented with non-essential amino acids derived from Eagles Medium, and which can further contain 0.6% bovine serum albumin, 20 μg heparin/mL and 5 mM caffeine. Alternately, oocytes can be fertilized in other medium containing non-essential amino acids such as the chemically defined medium described in the journal article entitled “Lowered Oxygen Tension and EDTA Improve Bovine Zygote Development In Chemically Defined Medium”, J. Anim. Sci. (1999), or the SOF medium described in the journal article “Successful Culture In-vitro of Sheep and Cattle Ova”, J. Reprod. Fertil. 30:493-497 (1972), each journal article hereby incorporated by reference.
[0030] After separating or sorting, sperm cells can be washed by centrifugation for about 10 min at 400 g in collection medium (typically Hepes-tyrode albumin lactate pyruvate medium supplemented with 2.0% bovine serum albumin) followed by suspension in the fertilization medium. Thawed, sorted sperm can be prepared by being centrifuged for 20 minutes at 700 g through a Percoll gradient (90%: 45%) for separation of live and dead sperm. The sperm pellet can then be washed with fertilization medium by centrifugation at 400 g for 10 minutes. Sperm can then be added to to the fertilization medium to give a concentration of 1-2 million/mL.
[00001] [TABLE-US-00001]
  Cleavage Stage of Oocytes Inseminated with Separated
  Sperm in Four Different Fertilization Media.
        % 2-cell   % 8-cell
  Media   No. oocytes   % cleavage   at 24 h   at 72 h
  Fert-TALP   168   76 6a   66
  Fert-TALP + neaa   176   7126b   67
  CDM   167   8975c   70
  SOF   145   8649d   69
a,b,c,dMeans with different superscripts differ (P < .05).
[0032] Now referring primarily to Table 1, as can be understood, oocytes inseminated with separated spermatozoa in fertilization medium containing non-essential amino acids according to the invention exhibit an increased rate of early development through at least the two cell stage.
[00002] [TABLE-US-00002]
  Embryonic Development and Blastocyst Quality
  Resulting From Fertilization in Four Different
  Fertilization Media (averaged over two culture media)
    % blastocysts/   % Grade 1
    oocyte   blastocysts/total
  Media   No. oocytes   Total   D7   blastocysts
  Fert-TALP   326   20   1752a,c
  Fert-TALP-aa   221   20   1768b  
  CDM   332   22   1861b,c
  SOF   321   21   1764b,c
a,b,cPercentages without common superscripts differ (P < .05)
dGrade 1 indicates blastocysts with a distinct inner cell mass suitable for embryo transfer.
[0034] Now referring primarily to Table 2, some embodiments of the invention in which oocytes are fertilized with sorted spermatozoa in fertilization medium containing supplemented non-essential amino acids can exhibit an enhanced quality of embryos. In embodiments of the invention in which oocytes were fertilized in tyrode albumin lactaate pyruvate (TALP) supplemented with non-essential amino acids derived from Eagles Medium, and further containing 0.6% bovine serum albumin, 20 μg heparin/mL and 5 mM caffeine there was a difference (P<0.05) in quality of embryos as compared to TALP without non-essential amino acids.
[0035] Presumptive zygotes can be removed from culture and placed in chemically-defined medium (CDM-1) as discussed in the Journal Animal Science, 78, 152-157 (2000), hereby incorporated by reference, for 6-7 hours after insemination and cultured for 65-66 hours. Embryos that cleaved were further cultured 96 hours in CDM-2 (further containing MEM essential and non-essential amino acids and 2.0 mM fructose) containing 0.12 IU insulin/mL. Blastocysts were morphologically graded according to the size of inner cell mass and stained with Giemsa to determine cell numbers on day 7 after insemination.
[0036] Now referring primarily to FIG. 4, the invention further involves a portable incubation system. Certain embodiments of the invention can comprise a straw (19) having an interior volume between about 0.1 mL and about 0.5 mL into which fertilization medium, and oocytes collected from a female mammal, can be transferred. While the straw (19) could be made of any material compatible with the fertilization medium and the collected oocytes, specific embodiments of the straw (19) can be made of plastic, such as or similar to an artificial insemination straw. The ends of plastic straws can be heat sealed after the fertilization medium and the oocytes are transferred inside.
[0037] The invention can further comprise an incubation element (20) configured to encapsulate the straw (19) or a plurality of straws inserted within. In some embodiments of the invention the incubation element (20) can be a glass tube having a single sealable aperture element. The aperture element (21) can be sealed with a cap (22), and in some embodiments the cap (22) and the tube can have spiral threads (23) that can be rotationally mated to close the incubation element (20).
[0038] After transfer of a straw (19) or a plurality of straws to the interior volume of the incubation element (20), incubation conditions can be established within. Typical incubation conditions within the interior volume of the incubation element can comprise an atmosphere of five percent carbon dioxide in air and a temperature of about 39° C. (37° C. to 41° C.). Once incubation conditions are established within the incubation element, the incubation element (20) can be sealed and the oocytes can then be transported within the incubation element (20).
[0039] In some embodiments of the invention, oocytes can be transported to a sperm cell separation facility where the incubation element (20) is unsealed, the straw (19) is unsealed and a plurality of sperm cells (15) from a population separated on the basis of bearing an X-chromosome or bearing a Y-chromosome can be transferred into the straw (19) containing the oocytes. With respect to some embodiments of the invention a concentration of separated sperm cells (15) can be established of between about 1 million to about 2 million/mL of the fertilization medium. The straw (19) containing the oocytes and spermatozoa in fertilization medium can then be resealed and transferred back into the incubation element (20). The incubation conditions can be re-established and the incubation element sealed. The incubation element (20) containing a straw or plurality of straws (19) can then be transported. During transport the oocytes can become fertilized. Upon arrival zygotes can be transferred from the straw for further culture.
[0040] With respect to certain embodiments of the invention, oocytes can first be inseminated with separated or unseparated spermatozoa in conventional 50 μl drops and loaded into a 0.25 mL straw or straws (19) within two hours after insemination. Straws (19) can be heat sealed and put into the incubation element (20). The open incubation element containing straws with inseminated oocytes can be equilabrated with 5% carbon dioxide in air at about 39° C. for at least one hour and then tightly capped and cultured under the same conditions for between about 18-20 hours.
[0041] Again referring primarily to Table 2, fewer oocytes (P<0.05) fertilized in Fert-TALP developed to the 2-cell stage by 24 hours than in any other media. Notably, the vast majority of oocytes (75%) fertilized in CDM medium cleaved to 2-cell stabe by this time. By 72 hours post-insemination, there was no difference between any of the media, possibly due to the long 8-cell stage cell cycle.
[0042] There was no difference between any of the media on rate of development to blastocysts. However, there was a significant difference in quality of embryos between Fert-TALP and Fert-TALP+non-essential amino acids.
[0043] Progression of early bovine embryonic development using separated sperm are similar to studies with in-vivo or in-vitro cleavage of oocytes fertilized with unseparated spermatozoa. In the cow the first in-vivo cleavage occurs at 24-28 hours following ovulation, and the first in-vitro cleavage tages place at 24-48 hours after insemination.
[0044] Earlier cleavage occurred with oocytes fertilized in CDM, SOF, and Fert-TALP+aa medium than in conventional Fert-TALP medium. This can be because CDM, SOF, and Fert-TALP+non-essential amino acids, all contain non-essential amino acids, which may play a role in how quickly spermatozoa penetrate oocytes, of in the length of the first cell cycle.
[0045] 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 the staining of spermatozoa, whether fresh spermatozoa or frozen-thawed spermatozoa, separation and isolation techniques which may be used with such stained spermatozoa, as well as devices to accomplish the staining, separation, isolation of such stained spermatozoa into X-chromosome bearing and Y-chromosome bearing populations, and the transporting of maturing oocytes and fertilized oocytes. In this patent application, the staining and separating techniques used with spermatozoa 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 the foregoing, all of these facets should be understood to be encompassed by this disclosure.
[0046] The discussion included in this patent application is intended to serve as a basic description. The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not fully explain the generic nature of the invention and may not explicitly show how each feature or element can actually be representative of a broader function or of a great variety of alternative or equivalent elements. Again, these are implicitly included in this disclosure. Where the invention is described in functionally-oriented terminology, each aspect of the function is accomplished by a device, subroutine, or program. 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 now be included.
[0047] Further, each of the various elements of the invention and claims may also be achieved in a variety of manners. This disclosure should be understood to encompass each such variation, be it a variation of an embodiment of any apparatus embodiment, a method or process embodiment, or even merely a variation of any element of these. Particularly, it should be understood that as the disclosure relates to elements of the invention, the words for each element may 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 that all actions may be expressed as a means for taking that action or as an element which causes that action. Similarly, each physical element disclosed should be understood to encompass a disclosure of the action which that physical element facilitates. Regarding this last aspect, as but one example, the disclosure of a “sorter” should be understood to encompass disclosure of the act of “sorting”—whether explicitly discussed or not—and, conversely, were there only disclosure of the act of “sorting”, such a disclosure should be understood to encompass disclosure of a “sorter” and even a “means for sorting”. Such changes and alternative terms are to be understood to be explicitly included in the description.
[0048] Additionally, the various combinations and permutations of all elements or applications can be created and presented. All can be done to optimize the design or performance in a specific application.
[0049] Any acts of law, statutes, regulations, or rules mentioned in this application for patent: or patents, publications, or other references mentioned in this application or any parent application for patent are hereby incorporated by reference. Specifically, U.S. application Ser. No. 10/433,191, filed May 29, 2003, International Application No. PCT/US01/45237, filed Nov. 29, 2001, U.S. Provisional Patent Application No. 60/253,787, filed Nov. 29, 2000 and U.S. Provisional Patent Application No. 60/253,785, filed Nov. 29, 2000, are hereby incorporated by reference including any figures or attachments.
[0050] In addition, as to each term used it should be understood that unless its utilization in this application is inconsistent with such interpretation, common dictionary definitions should be understood as incorporated for each term and all definitions, alternative terms, and synonyms such as contained in the Random House Webster's Unabridged Dictionary, second edition are hereby incorporated by reference. Finally, all references listed in an Information Disclosure Statement or other information statement filed with this application or in any parent applications are hereby appended and 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 with the patenting of this/these invention(s) such statements are expressly not to be considered as made by the applicant(s).
[0051] In addition, unless the context requires otherwise, it should be understood that the term “comprise” or variations such as “comprises” or “comprising”, 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 in countries such as Australia and the like.
[0052] Thus, the applicant(s) should be understood to have support to claim at least: i) each of the staining, separation, isolation, insemination, or fertilization procedures as herein disclosed and described, ii) the related methods disclosed and described, iii) similar, equivalent, and even implicit variations of each of these 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 that which is disclosed and described, vi) each feature, component, and step shown as separate and independent inventions, vii) the applications enhanced by the various systems or components disclosed, viii) the resulting products produced by such systems or components, ix) 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.
[0053] The claims set forth in this specification or in a parent specification are hereby incorporated by reference as part of this description of the invention, and the applicant expressly reserves the right to use all of or a portion of such incorporated content of such claims as additional description to support any of or all of the claims or any element or component thereof, and the applicant further expressly reserves the right to move any portion of or all of the incorporated content of such claims or any element or component thereof from the description into the claims or vice-versa as necessary to define the subject matter for which protection is sought by this application or by any subsequent continuation, division, or continuation-in-part application thereof, or to obtain any benefit of, reduction in fees pursuant to, or to comply with the patent laws, rules, or regulations of any country or treaty, and such content incorporated by reference shall survive during the entire pendency of this application including any subsequent continuation, division, or continuation-in-part application thereof or any reissue or extension thereon.


1. A method of in vitro fertilization of oocytes, comprising the steps of:
a) obtaining oocytes of a non-human mammal;
b) obtaining sperm cells of said non-human mammal separated into an X-chromosome bearing population or a Y-chromosome bearing population;
c) combining said oocytes of said non-human mammal in a fertilization medium supplemented with an amount of non-essential amino acids with said sperm cells of either said X-chromosome bearing population or said Y-chromosome bearing population;
d) establishing said plurality of oocytes with said sperm cells of said X-chromosome bearing population or said Y-chromosome bearing population in incubation conditions within an incubation element; and
e) fertilizing at least one of said oocytes of said non-human mammal with said sperm cells of said X-chromosome bearing population or said Y-chromosome bearing populations, said amount of non-essential amino acids sufficient to achieve an increased rate of development of the resulting at least one embryo.
2. The method of in vitro fertilization of oocytes of claim 1, wherein said oocytes fertilized with said sperm cells of said X-chromosome bearing population or said Y-chromosome bearing population in said fertilization medium containing non-essential amino acids achieves an increased rate of development through at least the two cell stage.
3. The method of in vitro fertilization of oocytes of claim 2, further comprising the step of entraining about 10 of said oocytes to about 15 of said oocytes in about 50 microliters of said fertilization medium.
4. The method of in vitro fertilization of oocytes of claim 3, wherein said fertilization medium is selected from the group consisting of: a chemically defined medium, SOF, Tyrode's medium supplemented with Eagles Medium, Tyrode's medium supplemented with 0.6 percent bovine serum albumin, 20 μg heparin per milliliter, and a concentration of 5 milli-molar caffeine, and combinations thereof.
5. The method of in vitro fertilization of oocytes of claim 1, wherein said incubation conditions comprise an atmosphere of 5 percent carbon dioxide in air at a temperature between about 37 degrees Centigrade and about 41 degrees Centigrade for a duration of about 18 hours to about 20 hours.
6. The method of in vitro fertilization of oocytes of claim 1, further comprising the step of establishing a concentration of said sperm cells of said X-chromosome bearing population or said Y-chromosome bearing population in said fertilization medium of about 1 million to about 2 million per milliliter of said fertilization medium.
7. The method of in vitro fertilization of oocytes of claim 1, further comprising the step of transferring said oocytes and said sperm cells into a straw.
8. The method of in vitro fertilization of oocytes of claim 7, wherein said straw has heat sealable aperture elements.
9. The method of in vitro fertilization of oocytes of claim 8, wherein said straw has an interior volume of about 0.25 milliliters.
10. The method of in vitro fertilization of oocytes of claim 7, wherein said incubation element has sealable aperture elements.
11. The method of in vitro fertilization of oocytes of claim 10, wherein said incubation element comprises a glass tube.
12. The method of in vitro fertilization of oocytes of claim 11, wherein said non-human mammal is selected from the group consisting of primates, bovids, ovids, equids, swine, and dolphins.

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