System And Method For Sorting Cells

  *US09365822B2*
  US009365822B2                                 
(12)United States Patent(10)Patent No.: US 9,365,822 B2
  et al. (45) Date of Patent:*Jun.  14, 2016

(54)System and method for sorting cells 
    
(75)Inventor: XY, LLC,  Navasota, TX (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 0 days. 
  This patent is subject to a terminal disclaimer. 
(21)Appl. No.: 13/764,408 
(22)Filed: Feb.  11, 2013 
(65)Prior Publication Data 
 US 2013/0150663 A1 Jun.  13, 2013 
 Related U.S. Patent Documents 
(60) .
Continuation of application No. 11/536,492, filed on Sep.  28, 2006 , which is a continuation of application No. 10/378,109, filed on Feb.  25, 2003, now Pat. No. 7,195,920 , which is a division of application No. 09/511,959, filed on Feb.  23, 2000, now Pat. No. 6,524,860 , which is a division of application No. 09/001,394, filed on Dec.  31, 1997, now Pat. No. 6,149,867 .
 
Jan.  1, 2013 C 12 N 5 0612 F I Jun.  14, 2016 US B H C Jan.  1, 2013 A 01 K 67 027 L I Jun.  14, 2016 US B H C Jan.  1, 2013 A 01 N 1 02 L I Jun.  14, 2016 US B H C Jan.  1, 2013 A 01 N 1 0226 L I Jun.  14, 2016 US B H C Jan.  1, 2013 A 61 D 19 02 L I Jun.  14, 2016 US B H C Jan.  1, 2013 A 61 D 19 04 L I Jun.  14, 2016 US B H C Jan.  1, 2013 C 12 N 15 873 L I Jun.  14, 2016 US B H C Jan.  1, 2013 C 12 Q 1 04 L I Jun.  14, 2016 US B H C Jan.  1, 2013 G 01 N 1 30 L I Jun.  14, 2016 US B H C Jan.  1, 2013 G 01 N 33 52 L I Jun.  14, 2016 US B H C Jan.  1, 2013 G 01 N 2015 149 L A Jun.  14, 2016 US B H C Jan.  15, 2015 Y 10 T 436 101666 L A Jun.  14, 2016 US B H C Jan.  15, 2015 Y 10 T 436 108331 L A Jun.  14, 2016 US B H C
(51)Int. Cl. C12N 005/071 (20100101); A01K 067/027 (20060101); A01N 001/02 (20060101); A61D 019/02 (20060101); C12N 015/873 (20100101); G01N 001/30 (20060101); G01N 033/52 (20060101); C12Q 001/04 (20060101); A61D 019/04 (20060101); G01N 015/14 (20060101)

 
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 5,796,112  A  8/1998    Ichie     
 5,798,276  A  8/1998    Haugland et al.     
 5,799,830  A  9/1998    Carroll et al.     
 5,804,143  A  9/1998    Leary     
 5,804,436  A  9/1998    Okun et al.     
 5,815,262  A  9/1998    Schrof et al.     
 5,819,948  A  10/1998    Van den Engh     
 5,824,269  A  10/1998    Kosaka et al.     
 5,831,723  A  11/1998    Kubota et al.     
 5,835,262  A  11/1998    Iketaki et al.     
 5,840,504  A  11/1998    Blecher     
 5,844,685  A  12/1998    Gontin     
 5,846,737  A  12/1998    Kang     
 5,866,344  A  2/1999    Georgiou     
 5,868,767  A  2/1999    Farley et al.     
 5,872,627  A  2/1999    Miers     
 5,873,254  A  2/1999    Arav     
 5,874,266  A  2/1999    Paisson     
 5,876,942  A  3/1999    Cheng et al.     
 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.     
 5,895,922  A  4/1999    Ho     
 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     
 6,042,249  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.     
 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     
 6,706,163  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    den Engh     
 6,941,005  B2  9/2005    Lary et al.     
 7,015,310  B2  3/2006    Remington et al.     
 7,094,527  B2  8/2006    Seidel et al.     
 7,105,355  B2  9/2006    Kurabayashi et al.     
 7,195,920  B2  3/2007    Seidel et al.     
 7,208,265  B1  4/2007    Schenk     
 7,221,453  B2  5/2007    Sharpe et al.     
 8,569,053  B2  10/2013    Seidel 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     
 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//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//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.     

 
 FOREIGN PATENT DOCUMENTS 
 
       BR       9704313                         4/1999      
       BR       9704313                         6/1999      
       CA       1029833                         4/1978      
       CA       1 250 808                         3/1989      
       CA       2113957       A1                1/1994      
       CA       2296324       A1                2/1999      
       CN       100998524                         7/2007      
       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 81                         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       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                5/2000      
       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                         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      
       JP       4126080 (A)                         4/1992      
       JP       4126081 (A)                         4/1992      
       JP       2007-170885                         7/2007      
       JP       2008-154957                         7/2008      
       SU       1056008                         11/1983      
       SU       1260778       A1                9/1986      
       WO       WO 84/01265       A1                4/1984      
       WO       WO 85/04014       A1                9/1985      
       WO       WO 88/07198                         9/1988      
       WO       WO 89/04470       A1                5/1989      
       WO       WO 89/04471       A1                5/1989      
       WO       WO 89/04472       A1                5/1989      
       WO       WO 90/13315       A1                11/1990      
       WO       9105236                         4/1991      
       WO       WO 92/08120       A1                5/1992      
       WO       WO 92/17288       A1                10/1992      
       WO       WO 93/10803                         6/1993      
       WO       9317322       A1                9/1993      
       WO       WO 94/22001       A1                9/1994      
       WO       WO 96/04542       A1                2/1996      
       WO       96/12171                         4/1996      
       WO       9612171       A3                4/1996      
       WO       WO 96/12171       A2                4/1996      
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       WO       WO 96/31764                         10/1996      
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       WO       WO 97/14785                         4/1997      
       WO       WO 97/29354       A1                8/1997      
       WO       WO 97/30338       A1                8/1997      
       WO       WO 97/35189       A1                9/1997      
       WO       WO 97/43620       A1                11/1997      
       WO       WO 98/34094       A1                8/1998      
       WO       WO 98/48259                         10/1998      
       WO       WO 98/57152       A1                12/1998      
       WO       WO 99/05504       A2                2/1999      
       WO       WO 99/33956       A1                7/1999      
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       WO       WO 99/44035                         9/1999      
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       WO       WO 01/28700       A1                4/2001      
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       WO       2007016090       A2                2/2007      

 OTHER PUBLICATIONS
  
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  Seidel et al., Insemination of heifers with sexed frozen or sexed liquid semen, Theriogenology, Jul. 1996, p. 400. *
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  Seidel et al., Training manual for embryo transfer in cattle, CHapter 4, 1991, p. 1-4 http://www.fao.org/docrep/004/t0117e/t0117e04.htm. *
  Fugger et al., “Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection.” Human Reproduction, 1998, vol. 13, No. 9, pp. 2367-2370.
  Klinc et al., “Reduction of Oxidative Stress in Bovine Spermatozoa During Flow Cytometric Sorting”, Reprod Dom Anim, 2007, 42, 63-67.
  Rath et al., “Improved quality of sex-sorted sperm: A prerequisite for wider commercial application” Theriogenology, 2009, 71, 22-29.
  US Office Action dated Jun. 14, 2013 in corresponding U.S. Appl. No. 11/536,492.
  HK Notice of Publication of the Registration and Grant of a Standard Patent dated Jul. 5, 2013 in corresponding HK patent application No. 10101116.5.
  AR Final Office Action dated Jun. 7, 2013 in corresponding AR Patent Application No. 20060103544.
  Chinese Office Action dated May 6, 2013 in corresponding CN Patent Application No. 200810128061.0.
  Japanese Office Action dated Apr. 17, 2013 in corresponding JP Patent Application No. 2010-139978.
  Mexican Notice of Allowance dated Mar. 5, 2013 in corresponding MX Patent Application No. MX/a/2012/006915.
  Chinese Notice of Allowance dated Jun. 6, 2012 in corresponding CN Patent Application No. 200810128062.5.
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  HK Grant of a Standard Patent dated Aug. 9, 2013 in related HK Patent Application No. 10101120.9. (3 pages).
  Brazilian Office Action dated Apr. 30, 2013 in related BR Patent Application No. PI 9816371-0. (6 pages).
  CN Notice of Allowance dated Aug. 2, 2013 in related CN Patent Application No. 200810128061.0. (3 pages).
  JP Final Rejection dated Aug. 6, 2013 in related JP Patent Application No. 2010-139978 (16 pages).
  US Office Action dated Sep. 11, 2013 in related U.S. Appl. No. 13/764,390. (10 Pages).
  EPO Extended Search Report dated Sep. 26, 2013, issued in related EP Patent Application No. 10185715.9 (8 pages).
  Johnson, et al. “Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting”, Biology of Reproduction, vol. 41, 1989, pp. 199-203, XP002103476.
  Hawk, et al.“Fertilization rates in superovulating cows after deposition of semen on the infundibulum near the uterotubal junction or after insemination with high numbers of sperm,” 1988, XP-002103478, Abstract, (1 page).
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  Sharpe, J.C., et al., “Advances in flow cytometry for sperm sexing”, Theriogenology, 71 (2009) pp. 4-10.
  US Office Action dated Aug. 12, 2014, issued in related U.S. Appl. No. 13/764,390 (10 pp).
  Uruguayan Office Action dated Aug. 18, 2014, issued in related UY Application No. 26669 (3 pp).
  US Petition for Inter Partes Review No. 2014-01161 filed Jul. 14, 2014, (43 pp).
  Declaration and Curriculum Vitae of Peter Lopez as filed in Inter Partes Review No. 2014-01161 dated Jul. 13, 2014 (75 pp).
  XY LLC's Opening Claim Construction Brief, XY LLC v. Trans Ova Genetics, LC, et al., Civil No. 1:13-cv-00876-WJM-BNB.
  CA Requisition by the Examiner issued on Dec. 17, 2014 for CA Application No. 2,823,259.
  JP Notice of Final Rejection issued on Nov. 27, 2014 for JP Application No. 2013-021330.
  Brazilian Office Action issued Oct. 1, 2004 for related BR application No. PI 9816371-0.
  EPO Intent to Grant issued Feb. 20, 2015 for related EP application No. 10185715.9.
  EPO Intent to Grant issued Mar. 18, 2015 for related EP application No. 10185761.3.
  EPO Intent to Grant issued Mar. 23, 2015 for related EP application No. 07008703.6.
  EPO Decision to Grant issued May 15, 2015 for related EP application No. 10185715.9.
  EPO Decision to Grant issued May 21, 2015 for related EP application No. 07008703.6.
  EPO Decision to Grant issued May 21, 2015 for related EP application No. 10185761.3.
  EP Office Action dated Oct. 3, 2014, issued in related EP Application No. 07008703.6 (3 pp).
  CL Office Action dated Apr. 29, 2014, issued in corresponding CL Application No. 2000-1766 (2 pp).
  JP Office Action dated Jun. 16, 2014, issued in corresponding JP Application No. 2013-021330 (13 pp).
  EP Examination Report issued Jan. 31, 2014, issued in corresponding EP application No. 10185715.9 (5 pp).
  EP Examination Report issued Feb. 17, 2014, issued in corresponding EP application No. 07008703.6 (6 pp).
  US Final Office Action dated Feb. 25, 2014, issued in corresponding U.S. Appl. No. 11/536,492 (8 pp).
  US Office Action dated Mar. 27, 2014, issued in corresponding U.S. Appl. No. 13/764,390 (8 pp).
  EP Examination Report issued Jan. 30, 2014 in corresponding EP application No. 10185761.3(5 pp).
  US Final Office Action issued Dec. 11, 2013, in corresponding U.S. Appl. No. 13/764,390 (7pp.).
  EPO Extended Search Report dated Sep. 26, 2013, issued in related EP Application No. 10185715.9 (8 pp).
  Canadian patent application No. 2,316,080; OA mailed Feb. 10, 2012, 9 total pages.
  Chinese patent application No. 200810128055.5; OA mailed May 3, 2012, 12 total pages.
  Chinese patent application No. 200810128055.5; OA mailed Mar. 26, 2013, 14 total pages.
  Chinese patent application No. 200810128058.9; OA mailed May 3, 2012, 8 total pages.
  Chinese patent application No. 200810128059.3; OA mailed May 3, 2012, 10 total pages.
  Chinese patent application No. 200810128059.3; OA mailed Mar. 26, 2013, 11 total pages.
  Chinese patent application No. 200810128060.6; OA mailed May 3, 2012, 12 total pages.
  Chinese patent application No. 200810128060.6; OA mailed Mar. 26, 2013, 10 total pages.
  Chinese patent application No. 200810128061.0; OA mailed May 3, 2012, 12 total pages.
  Chinese patent application No. 200810128058.9; OA mailed Mar. 26, 2013, 9 total pages.
  EP patent application No. 989865046.0; OA mailed Oct. 15, 2012, 13 total pages.
  Corresponding Argentine patent application No. 20060103544: OA mailed Jan. 17, 2013, 8 total pages.
  Corresponding Japanese Application No. 2010-139978; Office Action dated Sep. 11, 2012, 2 total pages.
  Corresponding Chinese Application No. 200810128061.0; Office Action dated Dec. 28, 2012, 7 total pages.
  Defendant's Disclosure of Invalidity Contentions, XY, LLC v. Trans Ova Genetics, L.C., Case No. 5:12-CV-208-OLG, U.S. Dist. Ct., W.D. Texas, dated Aug. 20, 2012, 45 total pages.
  Defendant's Answer, Affirmative Defenses and Jury Demand, XY, LLC v. Trans Ova Genetics, L.C., Case No. 5:12-CV-208-OLG, U.S. Dist. Ct., W.D. Texas, dated Aug. 1, 2012, 64 total pages.
  Plaintiff XY, LLC's Motions to Dismiss, XY, LLC v. Trans Ova Genetics, L.C., Case No. 5:12-CV-208-OLG, U.S. Dist. Ct., W.D. Texas, dated Aug. 28, 2012, 15 total pages.
  Reply in Support of XY, LLC's Motions to Dismiss, XY, LLC v. Trans Ova Genetics, L.C., Case No. 5:12-CV-208-OLG, U.S. Dist. Ct., W.D. Texas, dated Sep. 18, 2012, 31 total pages.
  Amblard. Method to Assess the Strength of Cell-Cell Adhesion Using a Modified Flow Cytometer; Chapter 7 in “Studying Cell Adhesion,” P. Bongrand, P.M. Claesson, A.S.G. Curtis, eds., Springer-Verlag, Berlin, 1994, pp. 93-108.
  Amblard, et al. New Chamber for Flow Cytometric Analysis Over an Extended Range of Stream Velocity and Application to Cell Adhesion Measurements; Cytometry, 1992, 13:15-22.
  Kay, et al. Experimental Findings on Gynecologic Cell Orientation and Dynamics for Three Flow Nozzle Geometries; J. of Histochemistry and Cytochemistry, Jul. 1977, vol. 25, No. 7, pp. 870-874.
  Lorton, et al., A New Antibiotic Combination for Frozen Bovine Semen: 2. Evaluation of Seminal Quality; Theriogenology, Mar. 1988, vol. 29, No. 3, pp. 593-607.
  Shin, et al. A New Antibiotic Combination for Frozen Bovine Semen: 1. Control of Mycoplasmas, Ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus; Theriogenology, Mar. 1988, vol. 29, No. 3, pp. 577-591.
  Watkins, et al. Analysis of the flow Cytometer Stain Hoechst 33342 on Human Spermatozoa; Molecular Human Reproduction, 1996, vol. 2, No. 9, pp. 709-712.
  Corresponding Argentine Application No. 20060103544; Office Action dated Aug. 21, 2012, 5 pages.
  Corresponding Chinese Application No. 200810128055.5; Office Action dated Sep. 19, 2012, 11 pages.
  Corresponding Chinese Application No. 200810128059.3; Office Action dated Sep. 19, 2012, 9 pages.
  Corresponding Chinese Application No. 200810128060.6; Office Action dated Sep. 19, 2012, 12 pages.
  Corresponding Chinese Application No. 200810128058.9; Office Action dated Sep. 19, 2012, 10 pages.
  Corresponding European Application No. 07 008 703.6; Office Action dated Sep. 7, 2012, 6 pages.
  Garner, et al. Effect of Semen Dilution on Bovine Sperm Viability as Determined by Dual-DNA Staining and Flow Cytometry. Journal of Andrology, May/Jun. 1997, vol. 18, No. 3, pp. 324-331.
  Januskauskas, et al. Effect of Cooling Rates on Post-Thaw Sperm Motility, Membrane Integrity, Capacitation Status and Fertility of Dairy Bull Semen Used for Artificial Insemination in Sweden. Theriogenology, Sep. 1999, 52(4), pp. 641-658.
  Corresponding German Patent Appl. No. 198 61 445.4; Office Action mailed Jul. 5, 2012, 12 total pages.
  Corresponding Chinese Patent Appl. No. 200810128056.X; Office Action mailed Jun. 6, 2012, 7 total pages.
  Opposition for EP 1 044 242 B1, dated Jul. 15, 2009, 39 total pages.
  Opposition, Translation; for EP 1 044 242 B1, dated Jul. 15, 2009, 37 total pages.
  Patentee Response to Opposition for EP 1 044 242 B1, dated Mar. 26, 2010, 21 total pages.
  Patentee Response with Amended Claims for EP 1 044 242 B1, 6 total pages.
  Opponents Reply for EP 1 044 242 B1, dated Sep. 9, 2011, 11 total pages.
  Opponents Reply, Translation; for EP 1 044 242 B1, dated Sep. 9, 2011, 11 total pages.
  Official Action—Decision for Rejection, issued on Oct. 10, 2011 in corresponding Chinese Application 200810128056.X 2 total pages.
  4th Office Action issued on Nov. 16, 2011 in corresponding Chinese Application No. 200810128062.5, 2 total pages.
  4th Office Action issued on Dec. 31, 2011 in corresponding Chinese application No. 200810128056.X, 2 total pages.
  Corresponding Chilean Application No. 1766-00; Office Action dated Aug. 26, 2011, 11 pages.
  Corresponding Chinese Application No. 200810128062.5; Office Action dated Jun. 30, 2011, 13 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.
  Parallel Canadian application No. 2,316,080 Office Action dated, Jun. 10, 2010, 6 pages.
  Parallel Chinese application No. 200810128062.5, Office Action dated, Oct. 15, 2010, 6 pages.
  Japanese Apellate Decision dated Jul. 23, 2010.
  Johnson, L.A. at al, “Sex Preselection in Rabbits: live births from X and Y sperm seperated by DNA and cell sorting,” Biology of Reproduction, vol. 41. 1989, pp. 199-203.
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  Correa, J.R. et al., “Quantitative and Qualitative Characteristics of Frozen-Thawed Bovine Spermatozoa . . . ,” Tohoku Journal of Exp. Med, vol. 181, pp. 267-274.
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  Mapletoft, R. J. et al., Superovulation in perspective, Bion che Animal Health, Dec. 2002.
  Bahr, G.F.et al., Considerations of volume, mass, DNA, and arrangement of mitochondria in the midpiece of bull spermatozoa, Experimental Cell Research 60 (1970) 338-340.
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  XY's Response to Trans Ova's Motion for Leave to File Reply Brief in Support of Its Motion or Leave to Serve Additional Request for Production. XY, LLC v. Trans Ova Genetics, L. C.. Case No. 1:13-cv-00876-WJM-BNB, US Dist. Ct., D. Colorado, dated Aug. 28, 2014, 3 pages total.
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  Defendant's Designations of Deposition Testimony. XY, LLC v. Trans Ova Genetics, L. C.. Case No. 1:13-cv-00876-WJM-BNB, US Dist. Ct., D. Colorado, dated Oct. 6, 2014, 9 pages total.
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     * cited by examiner
 
     Primary Examiner —Taeyoon Kim
     Assistant Examiner —Tiffany Gough
     Art Unit — 1651
     Exemplary claim number — 1
 
(74)Attorney, Agent, or Firm — Ryan Christensen; Hashim Rahman

(57)

Abstract

Improved flow cytometer system particularly adapted to use for sex-selected sperm sorting include enhanced sheath fluid and other strategies which minimize stress on the sperm cells, including a 2.9 percent sodium citrate sheath solution for bovine species and a hepes bovine gamete media for equine species. Improved collection systems and techniques for the process are described so that commercial applications of sperms samples as well as the resulting animals may be achieved.
11 Claims, 4 Drawing Sheets, and 4 Figures


[0001] This Application is a continuation of U.S. patent application Ser. No. 11/536,492, filed on Sep. 28, 2006, which is a continuation U.S. patent application Ser. No. 10/378,109, filed Feb. 25, 2003, now U.S. Pat. No. 7,195,920, which is a divisional of U.S. patent application Ser. No. 09/511,959, filed on Feb. 23, 2000, now U.S. Pat. No. 6,524,860, which is a divisional of U.S. patent application Ser. No. 09/001,394, filed on Dec. 31, 1997, now U.S. Pat. No. 6,149,867, each of which are hereby incorporated by reference.

I. BACKGROUND OF THE INVENTION

[0002] This invention relates generally to the field of sex selection in mammalian offspring. It is especially relevant to the aspect of low dose artificial insemination for creating the desired sex of offspring. Particularly, the invention relates to systems for sorting sperm via flow cytometry for sex-specific and low dose efforts at artificial insemination or the like.
[0003] For ages it has been desired to select the sex of specific offspring. Beyond obvious psychological aspects, the actual sex selection of mammalian offspring has significant economic consequences when one considers its application to food producing animals such as cattle as well as celebrated trophy animals such as horses and the like. This great desire has resulted in a significant variety of efforts to achieve sex-selected offspring. Probably the effort which has appeared most likely to achieve the desired results has been efforts at sorting and selecting between X and Y sperm prior to insemination.
[0004] One of the challenges that effort at sorting X and Y sperm has faced is the large numbers of sperm involved. In natural insemination sperm are produced in some species by the billions; in artificial insemination less, but still significantly large numbers of sperm are used. For instance, artificial insemination techniques commonly use ten million to five hundred million sperm (depending on species). Thus a significant number of sperm are necessary even in an artificial insemination environment.
[0005] Many methods have been attempted to achieve the separation of X- and Y-chromosome bearing sperm. These methods have ranged from magnetic techniques such as appears disclosed in U.S. Pat. No. 4,276,139 to columnar techniques as appears disclosed in U.S. Pat. No. 5,514,537 to gravimetric techniques as discussed in U.S. Pat. No. 3,894,529, reissue Pat. No. 32350, U.S. Pat. Nos. 4,092,229, 4,067,965, and 4,155,831. Electrical properties have also been attempted as shown in U.S. Pat. No. 4,083,957 as well as a combination of electrical and gravimetric properties as discussed in U.S. Pat. Nos. 4,225,405, 4,698,142, and 4,749,458. Motility efforts have also been attempted as shown in U.S. Pat. Nos. 4,009,260 and 4,339,434. Chemical techniques such as those shown in U.S. Pat. Nos. 4,511,661 and 4,999,283 (involving monoclonal antibodies) and U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,362, and 5,660,997 (involving membrane proteins), and U.S. Pat. Nos. 3,687,803, 4,191,749, 4,448,767, and 4,680,258 (involving antibodies) as well as the addition of serum components as shown in U.S. Pat. No. 4,085,205. While each of these techniques has been presented as if to be highly efficient, in fact at present none of those techniques yield the desired level of sex preselection.
[0006] At present, the only quantitative technique used to achieve the separation of X- and Y-chromosome bearing sperm has been that involving individual discrimination and separation of the sperm through the techniques of flow cytometry. This technique appeared possible as a result of advances and discoveries involving the differential dye absorption of X- and Y-chromosome bearing sperm. This was discussed early in U.S. Pat. No. 4,362,246 and significantly expanded upon through the techniques disclosed by Lawrence Johnson in U.S. Pat. No. 5,135,759. The Johnson technique of utilizing flow cytometry to separate X- and Y-chromosome bearing sperm has been so significant an advancement that it has for the first time made the commercial separation of such sperm feasible. While still experimental, separation has been significantly enhanced through the utilization of high speed flow cytometers such as the MoFlo7 flow cytometer produced by Cytomation, Inc. and discussed in a variety of other patents including U.S. Pat. Nos. 5,150,313, 5,602,039, 5,602,349, and 5,643,796 as well as international PCT patent publication WO 96/12171. While the utilization of Cytomation's MoFlo® cytometers has permitted great increases in speed, and while these speed increases are particularly relevant given the high number of sperm often used, certain problems have still remained. In spite of the almost ten-fold advances in speed possible by the MoFlo® flow cytometer, shorter and shorter sorting times have been desired for several reasons. First, it has been discovered that as a practical matter, the sperm are time-critical cells. They loose their effectiveness the longer they remain unused. Second, the collection, sorting, and insemination timings has made speed an item of high commercial importance. Thus, the time critical nature of the sperm cells and the process has made speed an essential element in achieving high efficacy and success rates.
[0007] Other problems also exist ranging from the practical to the theoretical. On the practical side, it has been desired to achieve sex-sorted sperm samples using inexpensive disposable components and substances. Also on the expense side, it has been desired to be able to achieve sorting (as well as collection and insemination) in as efficient a labor event as possible. Thus, for commercial production and success in this field, improvements which might only represent an increase in efficiency may still be significant. Related to the practical aspect of expense, is the practical aspect of the delicateness and sensitivity of the entire process. In this regard, it has been desired to simplify the process and make it as procedurally robust as possible so that operator error or skill can play an ever decreasing role.
[0008] In addition to the delicateness of the process, it has always been known that the sperm themselves are extremely delicate cells. While this factor at first glance seems like it might be considered easily understood, in fact, the full extent of the cells' sensitivities have not yet been fully explored. In the context of flow cytometry in general, most sorted cells or particles have often been spherical or otherwise physically able to withstand a variety of abuses. This is not the case for sperm cells. In fact, as the present invention discloses, the processing through normal flow cytometer techniques may, in fact, be unacceptable for cytometric sorting of sperm cells in certain applications. The sensitivities range from dilution problems and the flow cytometer's inherent need to isolate and distinguish each cell individually as well as the pressure and other stresses which typical flow cytometry has, prior to the present invention, imposed upon the cells or other substances that it was sorting. This may also represent a unique factor for sperm cells because it appears that even though the sperm cell may appear to pass through the flow cytometer and be sorted with no visually discernable side-effects, in fact, the cells themselves may have been stressed to the point that they perform less than optimally in the insemination process. Thus, an interplay of factors seems involved and has raised unusual problems from the perspective of sperm cell sorting and ultimate use for artificial insemination.
[0009] Another problem which has remained—in spite of the great advances achieved through the Johnson patent and related technology—is the fact that prior to the present invention it has been extremely difficult to achieve lower dosage insemination with sexed sperm. While historically, some achievement of low dose insemination has occurred, it has appeared to be more on a theoretical or laboratory environment rather than from environments which are likely to be experienced in or applicable to a commercial application. In this regard, the desire has not been merely to achieve low dose insemination but rather to achieve low dose insemination with pregnancy success rates which are comparable to existing unsexed, high dosage artificial insemination efforts. Thus, the advances achieved by the present inventors in both sexed and low dose artificial insemination represent significant advances which may, for the first time, make commercial applications feasible.
[0010] Another problem which has been faced by those in the industry—again, in spite of the great advances by the Johnson patent and related technology—is the fact that the problem itself, namely, artificial insemination with a high success rate is one of a statistical nature in which a multitude of factors seem to interplay. Thus, the solutions proposed may to some degree involve a combination of factors which, when thoroughly statistically studied, will be shown to be necessary either in isolation or in combination with other factors. Such a determination is further compounded by the fact that the results themselves vary by species and may be difficult to ascertain due to the fact that testing and statistical sampling on a large enough data base is not likely to be worth the effort at the initial stages. For these reasons the invention can also involve a combination of factors which may, individually or in combination, represent the appropriate solutions for a given application. This disclosure is thus to be considered broad enough so that the various combinations and permeations of the techniques disclosed may be achieved. Undiscovered synergies may exist with other factors. Such factors may range from factors within the sorting or flow cytometer steps to those in the collection as well as insemination steps. At present, studies have been primarily achieved on bovine species, however, it is not believed that these techniques will be limited to such species or, for that matter to only sperm cells. It appears that the techniques used may have application beyond just sperm cells into areas which involve either sensitive items to be sorted or merely minimization of the impacts of the stresses of flow cytometry upon the item sorted.
[0011] Interestingly, while the present invention takes an approach to minimize the impacts and stresses upon the sperm cells, others appear to have actually taken steps away from this direction by increasing pressures and demands for speed and other such performance. Essentially, the drive for low dose insemination and high speed processing may, in an individual or perhaps interrelated fashion have posed problems which limited one another. Thus, while there has been a long felt but unsatisfied need for high speed, low dose sexed insemination, and while the implementing arts and elements have long been available, prior to the present invention the advances or perhaps combinations of advances had apparently been overlooked by those skilled in the art. Perhaps to some degree they failed to appreciate that the problem involved an interplay of factors as well as peculiar necessities for the types of cells (sperm cells or perhaps species-specific sperm cells) involved in this field. Interestingly, as the listing of efforts earlier in this discussion shows, substantial attempts had been made but they apparently failed to understand the problem inherent in such an area as low dose, sexed insemination and had perhaps assumed that because the natural service event involves perhaps billions of sperm, there may have been physical limitations to the achievement of artificial insemination with numbers which are as many as four orders of magnitude less in number. Thus, it may not be surprising that there was to some extent an actual teaching away from the technical direction in which the present inventors went. Perhaps the results may even be considered unexpected to a degree because they have shown that sexed, low dose artificial insemination can be achieved with success rates comparable to those of unsexed, high dose artificial insemination. It might even be surprising to some that the techniques and advances of the present invention in fact combine to achieve the great results shown. While each technique could, in isolation, be viewed by some as unremarkable, in fact, the subtle changes appear to afford significant advances in the end result C whether considered alone or in combination with other subtle changes.
[0012] Thus, until the present invention the achievement of success rates for low dose, sexed artificial insemination has not been possible with levels of performance necessary or simplified procedures likely to be necessary to achieve commercial implementation. The present invention discloses techniques which permit the achievement of improved performances and thus facilitate the end result desired, namely, low dose, sexed artificial insemination on a commercial basis.

II. SUMMARY OF THE INVENTION

[0013] Accordingly, the present invention provides improved sheath and collector systems for sorting of sperm cells to determine their sex through a flow cytometer. The sheath fluid as typically used in a flow cytometer is replaced with a fluid which minimizes the stress on the sperm cells as they are sorted. Furthermore, the collection system is improved to minimize both the physical and chemical stress to which the sperm cells are subjected. Various techniques and substances are represented but as those skilled in the art will readily understand, various combinations and permutations can be used in the manner which may be optimized for performance based in the species, goals and other parameters involved in a specific processing application.
[0014] An object of the invention is thus to achieve better sorting for substances such as sperm cells. A goal is to minimize the impact the sorting function itself has on the cells or other sensitive items which may be sorted. A particular goal is to minimize the impact the sheath fluid imposes upon the cells and to potentially provide a sheath fluid which affirmatively acts to assist the cells in handling the various stresses involved. A parallel goal is to provide substances and techniques which are especially suited for sperm cells in general, for bovine sperm cells, for equine sperm cells, and for the separation of such sperm cells into X- and Y-chromosome bearing components. Similarly a goal is to minimize the impacts that the collection phase (e.g., after sorting) has upon the cells and to minimize the physical impact as well as chemical impacts on such sex sorted sperm cells. Thus a goal is to achieve as unaffected a sorted result as possible.
[0015] Another object of the invention is to achieve low dose, sorted insemination on levels and with success rates which are comparable to those of the typical unsexed, high dose artificial insemination. Thus the prior goals of minimizing the stress or potential damage upon the sperm cells is important. Sorting in a manner which affords both high speed and low stress sorting, and which is especially adapted for sperm cell sorting in a low dose context is an important goal as well. The goals of providing sheath and other fluids which do not negatively affect the fertility of the sperm and which are compatible with artificial insemination are also important.
[0016] Naturally further objects of the invention are disclosed throughout other areas of the specification and claims.

III. BRIEF DESCRIPTION OF DRAWINGS

[0017] FIG. 1 is a schematic diagram of a sorter system according to the present invention.
[0018] FIG. 2 is a diagram of the entrained cells in the free fall area of a typical flow cytometer.
[0019] FIG. 3 is a conceptual diagram showing differences as they roughly appear as a result of the present invention.
[0020] FIG. 4 is a diagram of the sorted cell stream as they are collected in the landing zone area.

IV. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0021] As will be seen, the basic concepts of the present invention can be combined and embodied in a variety of ways. The invention involves both improved flow cytometer systems as well as systems for the creation of sex-specific sperm samples which may be used in artificial insemination and the animals produced by such techniques. Furthermore, the techniques are disclosed in a general fashion so that they may be applied to specific systems and applications once the general principals are understood. While device enhancements are disclosed it should be understood that these enhancements not only accomplish certain methods but also can be varied and combined in a number of ways. Importantly, as to all of the foregoing, each of these facets should be understood to be encompassed by this disclosure.
[0022] As mentioned, the basic goal is that of separating the X-bearing sperm from the Y-bearing sperm. This is done in a manner which isolates the two types of sperm so that each can be separately packaged and dealt with. The isolation is preferably done through the use of flow cytometry. Flow cytometry in general is a technique which is well understood. For instance, the basic aspects of it are shown and discussed in a variety of patents to Cytomation, Inc. such as the U.S. Patents and other publications listed earlier. Each of these patents and the references cited therein, are incorporated by reference, thus those skilled in the art can easily understand the basic principles involved.
[0023] Essentially, flow cytometry involves sorting items, such as cells, which are provided to the flow cytometer instrument through some type of cell source. A conceptual instrument is shown in FIG. 1. The flow cytometer instrument includes a cell source (1) which acts to establish or supply cells or some other type of item to be analyzed by the flow cytometer. The cells are deposited within a nozzle (2) in a manner such that the cells are surrounded by a sheath fluid (3). The sheath fluid (3) is usually supplied by some sheath fluid source (4) so that as the cell source (1) supplies its cells, the sheath fluid (3) is concurrently fed through the nozzle (2). In this manner it can be easily understood how the sheath fluid (3) 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 nozzle (2) and exit at the nozzle orifice (5). By providing some type of oscillator (6) which may be very precisely controlled through an oscillator control (19), pressure waves may be established within the nozzle (2) and transmitted to the fluids exiting the nozzle (2) at nozzle orifice (5). Since the oscillator (6) thus acts upon the sheath fluid (3), the stream (7) exiting the nozzle orifice (5) eventually and regularly forms drops (8). Because the cells are surrounded by a sheath fluid environment, the drops (8) may contain within them individually isolated (generally) cells or other items.
[0024] Since the drops (8) generally contain isolated cells, the flow cytometer can distinguish and separate droplets based upon whether or not the appropriate cell or cells is/are contained within the drop. This is accomplished through a cell sensing system (9). The cell sensing system involves at least some type of sensor (10) which responds to the cells contained within each drop (8) as discussed at length in the seminal work (no pun intended) by Larry Johnson, namely, U.S. Pat. No. 5,135,759. As the Johnson patent explains for sperm cells, the cell sensing system (9) may cause an action depending upon the relative presence or relative absence of a particular dye which may be excited by some stimulant such as the laser exciter (11). While each type of sperm cell is stained by the dye, the differing length of the X-chromosome and the Y-chromosome causes different levels of staining, Thus, by sensing the degree of dye present in the sperm cells it is possible to discriminate between X-bearing sperm and Y-bearing sperm by their differing emission levels.
[0025] In order to achieve the ultimate separation and isolation of the appropriate cells, the signals received by sensor (10) are fed to some type of sorter discrimination system (12) which very rapidly makes the decision and can differentially charge each drop (8) based upon whether it has decided that the desired cell does or does not exist within that drop (8). In this manner the sorter discrimination system (12) acts to permit the electrostatic deflection plates (13) to deflect drops (8) based on whether or not they contain the appropriate cell or other item. As a result, the flow cytometer acts to sort the cells by causing them to land in one or more collectors (14). Thus by sensing some property of the cells or other items the flow cytometer can discriminate between cells based on a particular characteristic and place them in the appropriate collector (14). In the system presently used to sort sperm, the X-bearing sperm droplets are charged positively and thus deflect in one direction, the Y-bearing sperm 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] Referring to FIG. 2, the process can be even further understood. As shown in that figure, the nozzle (2) emits a stream (7) which because of the oscillator (6) (not shown in FIG. 2) forms drops (8). Since the cell source (1) (not shown in FIG. 2) may supply sperm cells (15) which have been stained according to the Johnson technique, the light stimulation by laser exciter (11) is differentially determined by sensor (10) so that the existence or nonexistence of a charge on each drop (8) as it separates from stream (7) can be controlled by the flow cytometer. This control results in positively charged, negatively charged, and uncharged drops (8) based upon their content. As shown in FIG. 2, certain drops are shown as deflected drops (16). These deflected drops (16) are those containing sperm cells (15) of the one or the other sex. They are then deposited in the appropriate collector (14) for later use.
[0027] One of the aspects of flow cytometry which is particularly important to its application for sperm sorting is the high speed operation of a flow cytometer. Advances have been particularly made by the flow cytometers available through Cytomation, Inc. under the MoFlo7 trademark. These flow cytometers have increased sorting speeds extraordinarily and have thus made flow cytometry a technique which is likely to make feasible the commercial application of sperm sorting (among other commercial applications). They act to achieve high speed sorting, that is at a speed which is notably higher than those otherwise utilized. Specifically, Cytomation's MoFlo7 flow cytometers act with oscillator frequencies of greater than about five kilohertz and more specifically can be operated in the 10 to 30 or even the 50 kilohertz ranges. Thus droplets are formed at very high frequencies and the cells contained within the sheath fluid environment can be emitted very rapidly from the nozzle (2). As a result, each of the components such as the nozzle (2) oscillator (6), and the like which make up and are part of a flow cytometer system result in a high speed cell sorter. In the application of a high speed cell sorter to the sorting of sperm cells, sorting at rates of greater than about 500 sorts per second is achieved. In fact, rates of sorting in the thousand and twelve hundred ranges have already been achieved through a high speed cell sorter. Importantly, it should be understood that the term “high speed” is a relative term such that as other advances in flow cytometry and specific applications are achieved, the aspect which is considered “high” may be varied or may remain absolute. In either definition, the general principle is that the sorting may occur at rates at which the parameters and physical characteristics of the flow cytometer are significant to the cells themselves when sorting particular cells such as sperm cells.
[0028] One aspect of high speed sorting which appears to come into play when sorting sperm cells is that of the pressures and other stresses to which the sperm cells are subjected within the flow cytometer. For instance, when operating at high speeds (and an alternative definition of “high speed”), flow cytometers can be operated at a pressure of 50 pounds per square inch and even 60 and higher pounds per square inch. These pressures may be considered high because they may result in effects upon the cells being sorted. The key as disclosed in the present invention for this facet is the fact that the stress thresholds of the particular cells are the determining factor. Additionally as further knowledge is gained it may be shown that the stress thresholds are a function of combined effects such as the particular species or the particular prior or subsequent handling of the cells. The key in this regard is that the stress imposed upon the cells can, in fact, alter their viability and their ability to achieve the desired result. In the pressure case, it may be that merely subjecting the sperm cells to a higher pressure as a result of the operation of the flow cytometer at that pressure may result in decreased performance of the cells. The present invention in one regard acts to minimize these stresses and thus results in greater efficacies as well as lower dosages as discussed later.
[0029] In considering the stress aspect of the cells, the present invention acts in a fashion which minimizes the stresses. These stresses can be minimized at any point in the over all cycle or process of collecting, sorting or even inseminating the animal. Importantly, the stress imposed by the handling of the cells within the flow cytometer appears significant for this application. In one embodiment of the invention, the sheath fluid is specifically selected so that it can serve in a coordinated fashion with both (or either) the pre-sort cell fluid environment or the post-sort cell fluid environment. While naturally it is possible to adjust either the pre- or post-sort fluids, in one embodiment the invention adjusts the sheath fluid (3) so that it imposes significantly less stress upon the cells than was previously accomplished. In one regard the invention is remarkable in that it removes the total focus from that of operation of the flow cytometer to a focus on handling and removing stress from the cells themselves. For instance, while it has been known to utilize fluids having a proper pH factor or osmoality, the present invention recognizes that there may be certain chemical compositions to which the cells may be hyper-responsive. These hyper-responsive chemical compositions may naturally vary based upon the cells or even the prior handling of the cells. Importantly at present it appears that for sperm cells certain metabolic chemical compositions such as citrate seem to prevent unusually high stresses upon the cells. Thus, the hyper-responsive chemical compositions can be defined as those to which the cells are particularly responsive in the context of their functionality and the then-existing handling techniques. As to sperm cells it appears that metabolic compositions, specifically citrate constancy for bovine sperm cells and hepes buffer constancy for equine sperm cells may be very important. Thus the present invention acts to minimize the changes through the type of operation or the selection of substances which may act as a means for minimizing the changes which the cells experience.
[0030] For the sheath fluid, a substance is selected according to one embodiment of the invention so that it may be chemically coordinated to prevent minimal changes. Thus, by selecting the appropriate sheath fluid not only in context of flow cytometry parameters, but rather also in context of the cell parameters themselves, the changes experienced by the cells and the over all result of the sorting can be enhanced. This is shown conceptually in FIG. 3. FIG. 3 shows some type of chemical factor (such as citrate or other factors) as it may exist throughout the various phases of the process. For instance, the four phases shown might represent the following: phase I may represent the existence of the cells within the cell source (1), phase II might show the existence of the cells as they are sorted in the sheath fluid environment, phase III might show the cells as they are collected after sorting and phase IV might show the reconstituted cells in a storage medium after sorting. These four phases as shown for the prior art may experience vastly different chemical factor environments. As shown conceptually, however, in the present invention the cells may experience very little change, most notably the dip or drop experienced between phases I and II may be virtually absent. This is as a result of the selection of the appropriate sheath fluid as mentioned above. Thus, as a result of being subjected to an appropriate sheath fluid, the cells in the present invention may experience a much lower level of stress.
[0031] One of the potential generalities that may exist with respect to this phenomenon is the fact that certain chemical compositions may represent more hyper-responsive chemical compositions than others. While naturally this may vary based upon the species of sperm, the handling, or even the type of cell involved, it appears that the viability of the cells for their intended purpose (here, artificial insemination) varies greatly, naturally or because of sorting or both, and so the cells exhibit a hyper-responsive character with respect to that chemical composition. By selecting certain metabolic chemical compositions, most notably citrates or chemicals which are within the citric acid cycle, great advances appear possible. Thus for the bovine sperm application, the sheath fluid (3) is selected and coordinated so that it presents about a 2.9 percent sodium citrate composition. Specifically, the 2.9 percent sodium citrate solution may be created as follows:
[0032] 1. Place 29.0 grams of sodium citrate dihydrate (Na3C6H5O7.2H2O) in a 1,000 ml volumetric flask
[0033] a. Dissolve sodium citrate in ¾ of water batch, then add water to volume.
[0034] 2. Add deionized or Nanopure water to make 1,000 ml final volume.
[0035] 3. Transfer to bottles and autoclave at 15 lbs pressure (245° F.) for at least 30 minutes
[0036] a. Autoclave solution using conditions to minimize evaporation (loose cover)
[0037] b. Be careful that water does not boil away.
[0038] 4. Cool slowly at room temperature.
[0039] 5. Store sealed in a 5° C. cold room.
[0040] Further, for a sheath fluid, the sodium citrate solution may be filtered.
[0041] 6. Filter with a 0.22 micron filter using aseptic techniques.
[0042] Interestingly, for equine sperm cells such a composition does not perform as well. Rather, it has been discovered that for equine sperm cells, a hepes buffered medium such as a hepes bovine gamete medium—particularly HBGM3 as previously created by J. J. Parrish for a bovine application—works well. This medium is discussed in the article “Capacitation of Bovine Sperm by Heparin”, 38 Biology of Reproduction 1171 (1988) hereby incorporated by reference. Not only is this surprising because it is not the same type of substance as is utilized for bovine sperm, but the actual buffer, originally was developed for a bovine application. Thus in the equine application the sheath fluid is selected which contains the hepes buffer. This solution may have a pH at room temperature of about 7.54 (pH at 39° C.=7.4) with the following composition:
[0043] 
[00001] [TABLE-US-00001]
 
    Chemical   Dry weight (g/500 ml)
 
 
  CaCl2   0.145  
  KCl2   0.115  
  MgCl2•6H2O   0.004  
  NaH2PO4•H2O   0.018  
    NaC1   2.525  
    NaPyruvate   0.011  
    Lactic Acid (60%)   1.84   ml
    HEPES   4.765  
  NaHCO3   0.420  
    BSA (fraction V)   3.0
 
[0044] One other aspect which may interplay in the present invention is the fact that the cells involved may experience unusual sensitivities. In one regard this may be due to the fact that sperm cells are in a class of cells which are non-repairing cells. That is, they do not have the ability to repair themselves and hence, they may need to be treated much more sensitively than is typical for flow cytometers or other handling equipment. Thus, it may be appropriate that the enhancement is particularly applicable when the flow cytometer acts to establish a source of sperm cells. Another potentially related aspect which may be unique to a class of cells such as sperm cells is the fact that their DNA is non-repairing, non-replicating, and non-transcribing. Either of these factors may come into play and so they may be relevant either individually or together. Thus, it may be that the teachings of the present invention apply to all gamete cells or even to viruses and the like which are non-repairing, non-translating, non-transcribing cells.
[0045] A separate aspect of the flow cytometer processing which may also be important is the fact of properly treating the cells both chemically and physically after they are sorted. As shown in FIG. 4, as the cells within drops (8) land in collector (14), it may be important that the container which makes up the collector be properly sized so that it acts as some means of avoiding an impact between the cells and the container itself. While it has been known to place an initial collector fluid (17) in the bottom of the container to collect the cells so that they do not hit the bottom of the container, it appears that a simple widening of the container to address variations in stream presentation as well as the inevitable splashing due to the impact of the cells into the container can be used to enhance the result. In one regard this can act as a cushioning element so that cells which may be mechanically delicate, that is, they may break or be damaged by an impact can be treated appropriately. Thus when the cytometer source establishes cells which are physically delicate cells as the cells to be sorted, it may be important to provide some type of cushioning element such as a wide collection tube for which the opening width (18) serves to position the walls of the container in a manner which avoids contact with the cells. Thus the tube does not present side walls so close that there is any significant probability of contact between those cells being sorted and the walls of the tube. In this manner, in addition to the collector fluid (17), it may be desirable to include a wide collection tube as well. Perhaps merely providing a wide opening to the container which serves as part of the collector (14) may be sufficient. For applications utilizing high speed sorting of sperm cells, it has been found that providing a container having an inner diameter opening of at least 15 millimeters is believed to be sufficient. Specifically when utilizing a 14 ml Falcon test tube in such an application, minimal physical damage to the cells as a result of the collector (14) has been discovered.
[0046] It should be noted that even the 14 ml Falcon test tube may not be optimum. Specifically, it is believed that designing a collection container which matches the geometry of the stream (that is, a “stream-matched container”) may be most optimal. This stream-matched container may have any or all of the following characteristics: a relatively wide orifice, an elliptically shaped orifice, a lesser height to width ratio than currently involved, an angled or otherwise coordinated presentation such as may present side walls which are parallel to the falling streams, and the like. It may also be desirable to provide a mounting element such as a movable element or medium like ball bearings or the like to permit variable orientation of the tube to match the falling stream desired to be collected. In addition, the physical characteristics for the class of containers such as the existing tube (described as a “Falcon-type” test tube) may include not only the width of the tube but also the material (such polystyrene to which the cells do not stick) out of which it is made and the like. (These material options are well known for the 14 ml Falcon tube.) Thus the container and it collection fluid may also serve as a cushioning element to minimize physical damage to the cells. It also can serve, by its size, to facilitate collection of adequate numbers of sperm without a significant dilution effect.
[0047] Another aspect of the collector fluid (17) can be the fact that it, too, may serve to minimize chemical stresses upon the cells. In one regard, since it may be important to provide a nutrient to the cells both before and after sorting, the collector fluid (17) may be selected so as to provide a coordinated level of nutrient so that the levels are balanced both before and after sorting. For bovine sperm in which a nutrient of egg yolk citrate is utilized at a two percent egg yolk level, it has been discovered that utilizing a six percent egg yolk citrate level (that is six percent egg yolk content in a citrate solution) provides good results. This is as result of the volumes existing before and after the sorting event. The collector fluid (17) may start (before sorting) with about 2 ml of volume. The sorting event may add about double this volume (ending at three times the initial starting volume) with very little egg yolk citrate in solution (due to clogging and other flow cytometer considerations). Thus, the end result in terms of the level of the amount of egg yolk citrate present may be equivalent to the starting result, namely, two percent egg yolk content in a citrate solution due to the volumes involved. Thus the collector fluid (17) may be selected so as to create an ending collector fluid environment which is balanced with the initial nutrient or other fluid environment. In this manner, it may serve to minimize the time and changed level of composition to which the cells are subjected. Naturally, these fluid environments may be presented within the flow cytometer or may exist at some other prior time, the important point being merely minimizing the stress to which the cells are subjected at any time in their life cycle. Furthermore, since the initial chemical substance content can be varied (for instance the percent egg yolk content in the citrate may be varied up or down), likewise the starting collection fluid environment or various volumes may also be varied so that the ending result is the same. Thus, prior to commencing the sorting process, the collector fluid exists with a six percent egg yolk content in the citrate solution and after completion of the sorting event the collector fluidCwith the sex-specific spermCmay result in a two percent egg yolk content in the citrate solution similar to the initial nutrient content.
[0048] Note that in later use these sperm cells may be treated to a 20% egg yolk content in the citrate fluid for other reasons, however these changes are not deemed to provide stress to the cells as they are merely a known part of the total insemination process. While naturally the levels may be varied as those skilled in the art readily understand, a 20% egg yolk citrate buffer may be constituted as follows:

I. Final Composition:

[0049] 80% sodium citrate solution (72 mM)
[0050] 20% (vol/vol) egg-yolk

II. Preparation for 1 Liter:

[0051] A. Sodium citrate solution
[0052] 1. Place 29.0 grams of sodium citrate dihydrate (Na3C6H5O7.2H2O) in a 1,000 ml volumetric flask
[0053] 2. Add deionized or Nanopure water to make 1,000 ml final volume.
[0054] 3. Transfer to bottles and autoclave at 15 lbs pressure (245° F.) for at least 30 minutes.
[0055] a. Autoclave solution using conditions to minimize evaporation (loose cover)
[0056] b. Be careful that water does not boil away.
[0057] 4. Cool slowly at room temperature.
[0058] 5. Store sealed in a 5° C. cold room.
[0059] B. Egg preparation
[0060] 1. Obtain fresh hen's eggs from a good commercial source.
[0061] 2. Wash the eggs free of dirt (do not use too much detergent) and rinse.
[0062] 3. Immerse eggs in 70% ethanol for 2-5 minutes.
[0063] 4. Remove eggs and allow to dry (or wipe dry) and store on a clean towel.
[0064] C. Preparation of extender
[0065] 1. Use sterile, clean glassware
[0066] 2. A-fraction (non-glycerol fraction)
[0067] a. Place 800 ml of 2.9% sodium citrate solution in a 1,000 ml graduated cylinder.
[0068] b. Antibiotic levels for the non-glycerol containing fraction (A-fraction) of the extender may be as follows:
[0069] I. Tylosin=100 μg/ml
[0070] ii. Gentamicin=500 μg/ml
[0071] iii. Linco-spectin=300/600 μg/ml
[0072] c. Add 200 ml of fresh egg-yolk as outlined below (Section D)
[0073] I. Mix very thoroughly.
[0074] d. This provides A-fraction extender based on 2.9% sodium citrate, with 20% egg-yolk and antibiotics at concentrations known to be non-toxic to bull sperm.
[0075] e. Extender can be stored overnight at 5° C.
[0076] f. Decant supernatant (upper 800 ml) the next day.
[0077] g. Warm to 37° C. prior to use the next day.
[0078] D. To add egg-yolk to a buffered solution, the following procedure works well.
[0079] 1. Wash egg and clean the eggs (see B above)
[0080] 2. Open egg and separate yolk from albumin using a yolk separator. Alternatively, pour yolk back and forth 2-3 times between the two half shells. Do not rupture the membrane around the yolk.
[0081] 3. Place the yolk onto a sterile piece of 15 cm filter paper.
[0082] 4. Hold the filter paper over the graduated cylinder containing buffer and squeeze the yolk (rupturing the membrane) and allow the yolk to run out of the folded filter paper into the cylinder. Typically about 12-15 ml of the yolk can be obtained from one egg.
[0083] Another aspect which may interplay in the various factors of the present invention is that of utilizing low dose amounts of sperm for artificial insemination or the like. Additional background on the aspect of sexed, artificial insemination may be found in “Prospects for Sorting Mammalian Sperm” by Rupert P. Amman and George E. Seidel, Jr., Colorado Associated University Press (1982) hereby incorporated by reference. As mentioned, natural insemination involves numbers of sperm on the order of billions of sperm. Typical artificial insemination is presently conducted with millions of sperm for bovine species and hundreds of millions of sperm for equine species. By the term “low dose” it is meant that the dosage of sperm utilized in the insemination event are less than one-half or preferably even less than about 10% of the typical number of sperm provided in a typical artificial insemination event. Thus, the term “low dose” is to be viewed in the context of the typical artificial insemination dosage or also as an absolute number. For bovine sperm where currently 1 to 10 million sperm are provided, a low dose process may be considered an absolute number of about 500,000 sperm or perhaps as low as 300,000 sperm or lower. In fact, through utilization of the techniques of the present invention, artificial insemination with good percentages of success has been shown with levels of insemination of sperm at 100,000 and 250,000 sperm (41% and 50%, respectively pregnancy rates). As shown in the article “Uterine Horn Insemination of Heifers With Very Low Numbers of Non-frozen and Sexed Spermatozoa” as published in 48 Theriogenology 1255 (1997) hereby incorporated by reference. Since sperm cells appear to display a sensitivity to dilution, these results may display particular interdependence on the utilization of low dose sperm samples with regards to various techniques of the present invention. The absolute numbers may be species dependent, for equine species, merely less than about ten, five, or even one million sperm may be considered a low dose process.
[0084] Another aspect which may be important is the fact that the sperm sexed through the present invention techniques is utilized in an artificial insemination system. Thus, when the collector (14) is used to provide sperm for artificial insemination the techniques of the present invention may be particularly relevant. Further, it is possible that the combination of both artificial insemination use and the use in a low dose environment may together create synergies which makes the various techniques of the present invention particularly appropriate. Naturally, the sexed sperm can be utilized not just in an artificial insemination mode, but in other techniques such as in vitro fertilization and the like.
[0085] The process of collecting, sorting, and eventually inseminating an animal through the use of flow cytometry involves a variety of steps. In the context of bovine insemination, first the semen is collected from the bull through the use of an artificial vagina. This occurs at rates of approximately 1.5 billion sperm per ml. This neat semen may be checked through the use of a spectrophotometer to assess concentration and may be microscopically evaluated to assure that it meets appropriate motility and viability standards. Antibiotics may then added. As a result the initial sample may have approximately 60 to 70 percent of the progressively motile sperm per ejaculate. For processing, a dilution through of some type TALP (tyrode albumin lactate pyruvate) may be used to get the numbers of sperm at a manageable level (for flow analysis) of approximately 100 million per ml. The TALP not only nurtures the sperm cells, but it may make them hyper-activated for the staining step. Prior to staining, in some species such as the equine species, centrifugation may be accomplished. Staining may be accomplished according to a multi-stained or single-stained protocol, the latter, the subject of the Johnson patent and related technology. The staining may be accomplished while also adjusting the extender to create the appropriate nutrient environment. In bovine applications this may involve adding approximately 20% egg yolk content in a citrate solution immediately after staining. Further, in staining the sperm cells, it has been discovered that by using higher amounts of stain than might to some extent be expected better results may be achieved. This high concentration staining may involve using amounts of stain in the tens of micro-molar content such as discussed in the examples below where 38 micro-molar content of Hoechst 33342 stain was used.
[0086] After adding the stain, an incubation period may be used such as incubating at one hour at 34° C. to hasten the dye uptake with concentrations at about 100 million sperm cells per ml. Filtration may then be accomplished to remove clumps of sperm cells and then dilution or extending may or may not be accomplished to the desired sort concentration of approximately 100 million sperm cells per ml may be accomplished. Sorting according to the various techniques discussed earlier may then be accomplished from which sperm cells may be recovered in the collection phase. As mentioned earlier, the collection may result in samples with approximately 2% egg yolk citrate concentrate content (for bovine species). This sample may then be concentrated to about 3-5 million sperm cells per ml through the use of centrifugation after which the sheath fluid and preserving fluid may be removed. A final extension may then be accomplished with either 20% egg yolk citrate or a Cornell Universal Extender or the like. The Cornell Universal Extender may have the following composition for 1000 ml:
[0087] 14.5 g sodium citrate dihydrate
[0088] 2.1 g NaHCO3
[0089] 0.4 g KCl
[0090] 3.0 g glucose
[0091] 9.37 g glycine
[0092] 0.87 g citric acid
[0093] For 20% egg-yolk
using 800 ml of above preparation and may include about 200 ml of egg-yolk composition.
[0094] After this last extending, 3 to 5 million sperm per ml (for bovine species) may result. This sample may then be cooled to slow the sperm's metabolism and to permit use over longer periods of time. In the equine species the sample may then be used in oviductal or other insemination processes as those skilled in the art well understand. In bovine sperm, the sample may be diluted yet one more time to the desired dosage level. It has been discovered that dilution may create an effect upon the sperm cell's viability and so it may be appropriate to avoid too large a level of dilution by providing a smaller sample. At present, low dosages of approximately 300,000 sperm per 0.184 ml may be achieved. Furthermore, it may be desirable to maintain a level of seminal plasma at approximately a five percent level, although the results of this requirement are, at present, mixed. The sperm cell specimen may then be placed in a straw for use in artificial insemination and may be transported to the cows or heifers to be inseminated.
[0095] In order to achieve conveniently timed artificial insemination, heifer or cow estrus may be synchronized using known techniques such as the utilization of prostaglandin F2α according to techniques well known in the art. This latter substance may be particularly valuable in that it has been reported to potentially achieve enhanced fertility in heifers as discussed in the article “Prostoglandin F2α—A Fertility Drug in Dairy Cattle?”, 18 Theriogenology 245 (1982) hereby incorporated by reference. While recent results have not maintained this premise, it may be that the present invention demonstrates its particular viability in situations of sexed, low dose insemination. For bovine species, artificial insemination may then be accomplished through the use of embryo transfer equipment with placement of the sperm cells deep within the uterine horns. This may be accomplished not at the peak moment as typically used in artificial insemination, but rather at a somewhat later moment such as 12 hours after that time since there is some possibility that fertility for sexed artificial insemination may occur slightly later. The utilization of embryo transfer equipment may be used because there may be high sensitivity of the uterine wall for such low dose, sexed inseminations.
[0096] Interestingly, rather than inseminating within the uterine body where such insemination are usually placed, by insemination deep within the uterine horn, better results may be achieved. Perhaps it is also surprising that the samples thus far studied have shown no difference between ipsi- and contra-lateral inseminations when accomplished deep within the uterine horn. By deep, it should be understood that the insertion is placed well into the uterine horn using the embryo transfer equipment. The fact that results do not appear significantly different using ipsi- and contra-lateral inseminations has led the present inventors to propose the use of insemination in both so that the process of identifying the appropriate uterine horn may no longer be needed.
[0097] As a result of the insemination, it is of course desired that an animal of the desired sex be produced. This animal may be produced according to the systems discussed earlier through the use of the sexed sperm specimen. It should also be understood that the techniques of the present invention may find application in other techniques such as laproscopic insemination, oviductal insemination, or the like.
[0098] As examples, the following experiments have been conducted. While not all use every aspect of the inventions described here, they do show the performance enhancements possible through differing aspects of the invention. Further, a summary of some experiments is contained in the article “Uterine Horn Insemination of Heifers With Very Low Numbers of Non-frozen and Sexed Spermatozoa” as referenced earlier. This article summarizes some of the data showing the efficacy of the present invention. As to the experiments, one has been conducted with sexed, unfrozen sperm cells with high success as follows:

Example 1

[0099] Angus heifers, 13-14 mo of age and in moderate body condition, were synchronized with 25 mg of prostaglandin F-2 alpha at 12-day intervals and inseminated 6-26 h after observed standing estrus. Freshly collected semen from three 14-26 mo old bulls was incubated in 38 μM Hoechst 33342 at 75×106 sperm/ml in a TALP medium for 1 h at 34° C. Sperm were sorted by sex chromosomes on the basis of epiflourescence from laser excitation at 351 and 364 nm at 150 mW using a MoFlo7 flow cytometer/cell sorter operating at 50 psi and using 2.9% Na citrate as sheath fluid. X chromosome-bearing sperm (˜90% purity as verified by resorting sonicated sperm aliquots) were collected at ˜500 live sperm/sec into 2-ml Eppendorf tubes containing 100 μl Cornell Universal Extender (CUE) with 20% egg yolk. Collected sperm were centrifuged at 600×g for 10 min and resuspended to 1.63×106 live sperm/ml in CUE. For a liquid semen unsexed control; Hoechst 33342-stained sperm were diluted with sheath fluid to 9×105 sperm/ml and centrifuged and resuspended to 1.63×106 progressively motile sperm/ml in CUE. Sexed semen and liquid control semen were cooled to 5° C. over 75 min and loaded into 0.25-ml straws (184 μl/straw). Straws were transported at 3 to 5° C. in a temperature-controlled beverage cooler 240 km for insemination 5 to 9 h after sorting. Sexed semen and liquid control semen were inseminated using side-opening blue sheaths (IMV), one half of each straw into each uterine horn (3×105 live sperm/heifer). As a standard control, semen from the same bulls had been frozen in 0.5-cc straws by standard procedures (mean 15.6×106 motile sperm/dose post-thaw), thawed at 35° C. for 30 sec, and inseminated into the uterine body. Treatments were balanced over the 3 bulls and 2 inseminators in a ratio of 3:2:2 inseminations for the sexed semen and two controls. Pregnancy was determined ultrasonically 31-34 days after insemination and confirmed 64-67 days later when fetuses also were sexed (blindly). Data are presented in the table.
[0100] 
[00002] [TABLE-US-00002]
 
    No.   No. Pregnant   No. Pregnant   No. female
  Treatment   Heifers bred   d31-34   d64-67   fetuses
 
  Sexed semen   45   20 (44%)   19 (42%)18 (95%)a
  Liquid control   28   15 (54%    15 (54%) 8 (53%)b
  Frozen control   29   16 (55%)   15 (52%)12 (80%)c
 
a,bSex ratios of values with different superscripts differ (P < 0.02).
[0101] Although the pregnancy rate with sexed semen was only 80% of controls, this difference was not statistically significant (>0.1). One pregnancy was lost by 64-67 d in each of the sexed and frozen control groups; 18 of 19 fetuses (95%) were female in the sexed group, and 20 of 30 (67%) were female in the control groups. The liquid semen control yielded a virtually identical pregnancy rate to the frozen semen control containing over 50 times more motile sperm (over 120 times more total sperm), demonstrating the efficacy of low-dose insemination into the uterine horns. We have altered the sex ratio in cattle significantly using flow cytometer technology and artificial insemination.
[0102] Similarly, an experiment was conducted with unsexed, unfrozen sperm cells and may be reported as follows:

Example 2

[0103] The objective was to determine pregnancy rates when heifers are inseminated with extremely low numbers of frozen sperm under ideal field conditions. Semen from three Holstein bulls of above average fertility was extended in homogenized milk, 7% glycerol (CSS) extender plus 5% homologous seminal plasma to 2×105, 5×105 or 10×106 (control) total sperm per 0.25 ml French straw and frozen in moving liquid nitrogen vapor. Semen was thawed in 37° C. water for 20 sec. Holstein heifers 13-15 mo of age weighing 350-450 kg were injected with 25 mg prostaglandin F-2-alpha (Lutalyse7) twice at a 12-day interval and inseminated with an embryo transfer straw gun and side-opening sheath, half of the semen deep into each uterine horn 12 or 24 h after detection of estrus. The experiment was done in five replicates over 5 months, and balanced over two insemination technicians. Ambient temperature at breeding was frequently −10 to −20° C., so care was taken to keep insemination equipment warm. Pregnancy was determined by detection of a viable fetus using ultrasound 40-44 days post-estrus and confirmed 55-62 days post-estrus; 4 of 202 conceptuses were lost between these times. Day 55-62 pregnancy rates were 55/103 (53%), 71/101, (70%), and 72/102 (71%) for 2×105, 5×105 and 10×106 total sperm/inseminate (P<0.1). Pregnancy rates were different (P<0.05) among bulls (59, 62, and 74%), but not between technicians (64 and 65%) or inseminations times post-estrus (65% for 12 h and 64% for 24 h, N=153 at each time). With the methods described, pregnancy rates in heifers were similar with 5×105 and 10×106 total sperm per inseminate.
[0104] Prior experiment has also been conducted on sexed, unfrozen sperm cells and may be reported as follows:

Example 3

[0105] Semen was collected from bulls at Atlantic Breeders Cooperative, diluted 1:4 with a HEPES-buffered extender+0.1% BSA, and transported 160 km (−2 HR) to Beltsville, Md. where it was sorted at ambient temperature by flow cytometry into a TEST yield (20%) extender using methods described previously (Biol Reprod 41:199). Sorting rates of up to 2×106 sperm of each sex per 5-6 h at −90% purity were achieved. Sperm were concentrated by centrifugation (300 g for 4 min) to 2×106 sperm/ml. Some sperm were sorted into extender containing homologous seminal plasma (final concentration, 5%). Sorted sperm were shipped by air to Colorado (−2,600 km) and stored at either ambient temperature or 5° C. (cooled during shipping over 6 hr in an Equitainer, an insulated device with an ice-containing compartment). Heifers or dry cows detected in estrus 11 to 36 h earlier were inseminated within 9 to 29 h of the end of the sperm sorting session. Sperm (1 to 2×105 in 0.1 ml) were deposited deep in the uterine horn ipsilateral to the ovary with the largest follicle as determined by ultrasound at the time of insemination.
[0106] None of 10 females became pregnant when inseminated with sperm shipped and stored at ambient temperature. Of 29 females inseminated with sperm cooled to 5° C. during shipping, 14 were pregnant at 4 weeks of gestation, and 12 (41%) at 8 weeks. Eleven of the 22 inseminated within 10 h of the end of sorting were pregnant at 8 weeks, but only 1 of 7 inseminated 17-24 h after sorting was pregnant. There was no significant effect of adding seminal plasma. One of the 12 fetuses was not of the predicted sex, one was unclear, and 10 were of the predicted sex, as determined by ultrasonography at 60-70 days of gestation.
[0107] Subsequently, 33 additional heifers were inseminated with 0.05 ml (semen extended as described above) into each uterine horn without using ultrasonography; only 3 were pregnant 4 weeks after insemination, and only 1 remained pregnant at 8 weeks. However, different bulls were used from the previous group, and all inseminations were done 18-29 h post-sorting. An additional 38 heifers were inseminated similarly (˜22 h post-sorting) 200 km from our laboratory with sorted sperm from another bull; none of these was pregnant 8 weeks after insemination.
[0108] To summarize, it is possible to achieve pregnancies in cattle via artificial insemination of sperm sorted for sex chromosomes by flow cytometry, and the sex ratio of fetuses approximates that predicted by reanalysis of sorted sperm for DNA content (90%). However, pregnancy rates varied greatly in these preliminary experiments which required shipping sperm long distances. Fertility decreased drastically by 17 h post-sorting, but there was some confounding because different bulls were used at the different times. Further studies are needed to determine whether variation observed in pregnancy rates was due to bull differences, insemination techniques, interval between sorting and insemination, or other factors.
[0109] Finally, an experiment also has been conducted with unsexed, unfrozen sperm cells and may be reported as follows:

Example 4

[0110] The objective was to determine pregnancy rates when heifers were inseminated with very low numbers of sperm under ideal experimental conditions. Semen from three Holstein bulls was extended in Cornell Universal Extender plus 5% homologous seminal plasma to 1×105 or 2.5×105 sperm per 0.1 ml; 2.5×106 total sperm per 0.25 ml was used as a control. Fully extended semen was packaged in modified 0.25 ml plastic French straws to deliver the 0.1 or 0.25 ml inseminate doses. Semen was cooled to 5° C. and used 26-57 h after collection. Holstein heifers 13-15 mo of age weighing 350-450 kg were injected with 25 mg prostaglandin F-2 alpha (Lutalyse7) at 12-day intervals and inseminated with an embryo transfer straw gun and side-opening sheath into one uterine horn 24 h after detection of estrus. Insemination was ipsilateral to the side with the largest follicle determined by ultrasound 12 h after estrus; side of ovulation was verified by detection of a corpus luteum by ultrasound 7-9 days post-estrus. Pregnancy was determined by detection of a fetus by ultrasound 42-45 days post estrus. The experiment was done in four replicates and balanced over three insemination technicians. Side of ovulation was determined correctly in 205 of 225 heifers (91%); surprisingly, pregnancy rates were nearly identical for ipsilateral and contralateral inseminates. Pregnancy rates were 38/93 (41%), 45/87 (52%), and 25/45 (56%) for 1×105, 2.5×105 and 2.5×106 sperm/-inseminate (P>0.1). There was a significant difference in pregnancy rate (P<0.05) among technician, but not among bulls. With the methods described, it may be possible to reduce sperm numbers per inseminate sufficiently that sperm sorted by sex with a flow cytometer would have commercial application.
[0111] As mentioned and as can be seen from the various experiments, the field is statistically based and thus a variety of additional experiments may be conducted to show the appropriate combination and limitation strategies. Thus synergies among various affects will further be identified, such as instances in which the dye effects and combined dye effects with laser excitation may be studied.
[0112] The discussion included in this application is intended to serve as a basic description. The reader should be aware that the specific discussion may not explicitly describe all embodiments possible; many alternatives are implicit. It also may not 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 device-oriented terminology, each element of the device implicitly performs a function. Apparatus claims may not only be included for the device 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 may be submitted. It should be understood that a variety of changes may be made without departing from the essence of the invention. Such changes are also implicitly included in the description. They still fall within the scope of this invention. A broad disclosure encompassing both the explicit embodiment(s) shown, the great variety of implicit alternative embodiments, and the broad methods or processes and the like are encompassed by this disclosure.
[0113] In addition, 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. As but one example of this aspect, the disclosure of a “collector” should be understood to encompass disclosure of the act of “collecting”—whether explicitly discussed or not—and, conversely, were there only disclosure of the act of “collecting”, such a disclosure should be understood to encompass disclosure of a “collector.” Such changes and alternative terms are to be understood to be explicitly included in the description.
[0114] Any references mentioned in the application for this patent as well as all references listed in any information disclosure filed with the application are hereby incorporated by reference. In addition, the table of references as presented below are hereby incorporated by reference. However, to the extent statements 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).
[0115] 
[00003] [TABLE-US-00003]
 
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  4,155,831   May 22, 1979   Bhattacharya
  4,191,749   Mar. 04, 1980   Bryant
  4,225,405   Sep. 30, 1980   Lawson
  4,276,139   Jun. 30, 1981   Lawson
  4,339,434   Jul. 13, 1982   Ericsson
  4,362,246   Dec. 07, 1982   Adair
  4,448,767   May 15, 1984   Bryant
  4,511,661   Apr. 16, 1985   Goldberg
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  5,346,990   Sep. 13, 1994   Spaulding
  5,371,585   Dec. 06, 1994   Morgan et al.
  5,439,362   Aug. 08, 1995   Spaulding
  5,466,572   Nov. 14, 1995   Sasaki et al.
  5,483,469   Jan. 09, 1996   Van den Engh et al.
  5,514,537   May 07, 1996   Chandler
  5,589,457   Dec. 31, 1996   Wiltbank
  5,602,039   Feb. 11, 1997   Van den Engh
  5,602,349   Feb. 11, 1997   Van den Engh
  5,660,997   Aug. 26, 1997   Spaulding
  5,690,895   Nov. 25, 1997   Matsumoto et al.
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[0116] 
[00004] [TABLE-US-00004]
 
  FOREIGN PATENT DOCUMENTS
    DOCUMENT NO   DATE   COUNTRY
 
    WO 96/12171   10/13/95   United States
    WO 98/34094   06/08/98   NZ
    WO 99/05504   07/24/98   US
    WO 99/33956   08/07/99   US
    WO 99/38883   05/08/99   US
    WO 99/42810   26/08/99   US
    WO 00/06193   10/02/00   US
 
[0117] 
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Claims

1. A method of producing at least one sexed embryo comprising:
producing a stream containing sperm cells, wherein the stream comprises sperm cells from a cell source surrounded by sheath fluid, wherein the sheath fluid surrounding the sperm cells includes an amount of citrate;
identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream;
collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid; and
fertilizing at least one egg with the collected sperm to form at least one sexed embryo.
2. The method of claim 1, wherein the at least one egg is fertilized in vitro with the collected sperm.
3. The method of claim 1, wherein the citrate comprises a chemical in the citric acid cycle.
4. The method of claim 1, wherein the at least one egg is fertilized in vivo.
5. The method of claim 4, wherein the step of fertilizing in vivo further comprises the step of delivering sperm cells into both uterine horns of a uterus.
6. The method of claim 5, the step of delivering sperm cells into both uterine horns further comprises inserting said sperm cells approximately between 12 to 24 hours inclusive after detecting estrus.
7. The method of claim 1, wherein the steps of producing a stream containing sperm, identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream, and collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid further comprises:
supplying sperm cells to a nozzle from the cell source;
creating a sheath fluid environment in the nozzle in which sperm cells are surrounded by the sheath fluid, wherein the sheath fluid environment comprises a citrate;
producing a stream containing the sperm cells surrounded by the sheath fluid;
sensing a property of the sperm cells in the stream, wherein said property corresponds to the presence of an X-chromosome or a Y-chromosome;
identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream; and
collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid.
8. The method of claim 7, wherein the sperm cells are present in a pre-sort environment prior to being supplied to the nozzle and wherein the sperm cells are present in a post-sort environment at collection.
9. The method of claim 8, wherein the pre-sort and/or the post-sort environments are coordinated with the sheath fluid environment.
10. The method of claim 9, wherein the pre-sort and/or post-sort environments comprise the same concentration of citrate as the sheath fluid environment.
11. A method of producing at least one sexed embryo comprising:
producing a stream containing sperm cells, wherein the stream comprises sperm cells from a cell source surrounded by sheath fluid, wherein the sheath fluid surrounding the sperm cells includes a citric acid;
identifying X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in the stream;
collecting X-chromosome bearing sperm cells and/or Y-chromosome bearing sperm cells in at least one collector having a collector fluid which includes a citric acid; and
fertilizing at least one egg with the collected sperm cells to form at least one sexed embryo.
*****

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