Este documento describe el protocolo para la congelación y descongelación de embriones bovinos. Explica los principios de criobiología, los componentes del medio de congelación, el proceso de congelación que incluye la clasificación de embriones, la adición de crioprotector, la siembra y la congelación controlada, y el proceso de descongelación y remoción del crioprotector. También discute los beneficios de la congelación de embriones y las enfermedades que no se transmiten a través de la transferencia de emb

1. Reuben J. Mapletoft* Department of Large Animal Clinical Sciences, WCVM, University of Saskatchewan *Bioniche Animal Health, Belleville, ON, Canada CONGELACION DE EMBRIONES

2. CONGELACION DE EMBRIONES

8. - 5 to - 7 °C - 30 to - 35 °C

14. Cell Volume Changes with Addition and Removal of Cryoprotective Agents (Mazur) Diluent Added Influx Efflux 1 Relative Cell Volume Time in Permeating Additive 0

16. Cell Volume Changes with Addition and Removal of Cryoprotective Agents (Mazur) Diluent Added Influx Efflux 1 Relative Cell Volume Time in Permeating Additive 0

17. Protocolo de Congelación

21. Lavado de embriones

24. BOVINE EMBRYOS: EXAMPLES OF DEVELOPMENTAL STAGE AND QUALITY Page 1

25. BOVINE EMBRYOS: EXAMPLES OF DEVELOPMENTAL STAGE AND QUALITY Page 2

26. BOVINE EMBRYOS: EXAMPLES OF DEVELOPMENTAL STAGE AND QUALITY Page 3

27. Calidad y Estadío de los Embriones Respuesta Posible a la Congelación Stroud y Leibo, 1995

28. 1333 Total 39,66 23/58 c Blastocisto expandido 52,03 179/344 bc Blastocisto 59,39 275/463 a Blastocisto temprano 55,40 0,004 259/468 ab Mórula % Valor P P/total Estado embrión

29. 1333 Total 33,33 11/33 b 3 49,23 32/65 ab 2 56,11 0,01 693/1235 a 1 % Valor P P/total Calidad Embrión

31. Cryoprotector Embryo Cotton plug Plug/label E511 SM010769 SRN 5G DT 1

38. Cryoprotector Embryo Cotton plug Plug/label E511 SM010769 SRN 5G DT 1

54. Cell Volume Changes with Addition of a Nonpermeating Cryoprotective Agent 1 Relative Cell Volume Time in Nonpermeating Additive 0 Added Removed Sucrose

58. Transferencia Directa de Embriones Bovinos

59. Cell Volume Changes with Addition and Removal of Glycerol vs Ethylene Glycol Diluent Added Influx Efflux 1 Relative Cell Volume Time in Permeating Additive 0

62. Comparison of Glycerol and Ethylene Glycol Pregnancy Rates % Preg. % Preg. References No. No. Leibo & Mapletoft (1998) 59.2 11376 58.0 1609 Holland Gen. (unpub.) 51.8 228 55.3 838 Arreseigor et al. (1998) 47.4 97 45.9 423 Looney et al. (1996) 50.0 56 69.6 56 Hinshaw et al. (1996) 59.6 780 56.2 185 Lange (1995) 58.2 189 48.9 92 Ethylene Glycol Glycerol

63. < Continued > 55.0 58.6 13331 4099 Total Em Tran (unpub.) (domestic transfers) 51.2 387 52.6 671 Em Tran (unpub.) (split donors, exported to Holland) 51.8 218 53.8 225 References % Preg. No. % Preg. No. Ethylene Glycol Glycerol

64. 1997 Census of Cattle Embryos Frozen and Transferred in Canada and the USA (Leibo and Mapletoft, 1998) 55.4 87.6 % of Total 58.5 15,433 56.9 12,411 USA 59.2 11,376 58.0 1,609 Canada % Pregnant No. Frozen % Pregnant No. Frozen Country Ethylene Glycol Glycerol

66. 100 90 80 70 60 50 40 Pregnancy (%) Seeding Temperatures of Direct Transfer Embryos Seeding Temperature ( o C) -5 -4 -6 -7 -8

67. 100 90 80 70 60 50 40 Pregnancy (%) Cooling Rate of Direct Transfer Embryos Cooling Rate ( o C/min) from -7 o C to -33 o C 0.4 0.5 0.6

69. Embryo Transfer in Canada in 2002 Embryos transferred fresh 27,135 Embryos transferred frozen 27,511 Direct transfer 27,606 (99%)

70. Effect of Thaw Method on Survival of Frozen Bovine IVF Embryos a,b Values differ significantly (P< 0.01) 295 (66) b 333 (74) b 447 Water 293 (63) b 248 (75) b 466 Air > Water 226 (47) a 262 (55) a 477 Air Hatched Blastocysts after 72 h (%) Blastocysts after 24 h (%) No. Thawed Thaw Method

71. Protocolo de Congelación

73. Direct Transfer Straw Ethylene glycol Embryo Cotton plug Plug/label E511 SM010769 SRN 5G DT 1

76. Pajuela de Transferencia Directa Etilenglicol 1,5 M

78. Trypsin for sanitary washing of embryos as specified by the IETS

85. Pajuela de Transferencia Directa Etilenglicol 1,5 M

87. Effect of Time Between Flushing and Freezing on Pregnancy Rate (Embryos frozen at Em Tran & thawed at Holland Genetics) (Hasler, 2001) 55.7 106 151 - 180 53.0 264 121 - 150 56.2 507 91 -120 56.0 722 61 - 90 56.0 418 31 - 60 54.1 85 0 - 30 % Pregnant No. Transfers Time (min)

93. 100 90 80 70 60 50 40 Pregnancy (%) Warming in Air of Direct Transfer Embryos Warming Time (sec) in Air 4 2 6 8 10

96. Peres Coelho et al., 2007 1122 Total 51,22 42/82 >360 segundos 56,79 372/655 >180≤360 segundos 55,84 0,42 251/385 ≤ 180 segundos % Valor P P/total Tiempo TETF

97. a b b a Estadio de desarrollo del embrión- Embriones congelados con etilenglicol

98. Embriones Brahman vs Bos Taurus (Looney et al., 2004) 57.7 542 940 55.5 197 355 Totales 43.7 48 98 203 30 20 67 Brahman 57.2 54 124 231 65 67 103 Sintéticas con Brahman 59.7 61 80 131 58 61 105 Continentales 63.5 64 240 375 61 49 80 Británicas % % Pre. N % Pre. N Total Etilenglicol Glicerol Biotipo

99. Estadio y Preñez (Looney et al., Therio 1996; 45:170) 14.0 2 14 7 30.4 7 23 6 25.0 4 16 5 58.8 30 51 4 % Preñadas N Estadio

100. Porcentajes de preñez con embriones congelados o refrigerados por 24-30 h y transferidos en receptoras cruzas Bos Indicus. Tribulo et al., 2001 Grado 1 Grado 2 Total Congelados 27/49 (55,1) 7/20 (35,0) a 34/69 (49,3) Refrigerados 21/36 (58,3) 7/13 (53,8) b 28/49 (57,1)

101. Refrigerated n=131 % Frozen-Thawed DT n=252

102. a ab ab b Operador

103. a b a Calificación de la Transferencia

104. RECEPTORAS TRANS . PREÑADAS % PREÑEZ 177 100 56.5% 181 121 67% 358 221 62% Grupo control Grupo FLUNIXIN Total Análisis Estadístico: efectos significativos del grupo tratado con Meglumina de Flunixin (p= 0.0458, Chi cuadrado) Utilización de inhibidores de la producción de prostaglandinas para mejorar los porcentajes de gestación en la transferencia embrionaria (Meglumina de Flunixin) D.Ezcurra, L. Guller Frers, E.V. Lopez

105. The effect of Banamine (flunixin meglumine) on pregnancy rate of embryo transfer recipients ab P<0.02 ( * Schrick, 2003; ** Burry, unpublished ) Banamine Controls 65.4 65.3 b % preg. 260 1,300 No. trans. 66.2 245 Burry ** 60.2 a 797 Schrick * % preg. No. trans. Study

106. Perdidas Embrionarias y Fetales

107. Embriones congelados en Etilenglicol n=252

108. Abortaron: 9 3.6% Paridas: 123 48.8% Terneros: 127 50.4% Muertos al Nac.: 7 2.8% Muertos: 1 0.4% TOTAL: 119 47.2%

109. Porcentaje Acumulativo de Preñez con 60 Días de Entore post ET

110. TERNERA BRANGUS PP NACIDO POR TE RECEPTORA

113. Procedures  Basal embryo holding medium  Sucrose or galactose  Need four media • Embryo holding medium • “Low” concentration EG • High concentration EG • Dilution medium

114. Van Wagtendonk et al., 1997  A thorough study  Used 0.25-ml straws – DT  Used glycerol – in straw dilution with 1 M sucrose  Pregnancy rates • Vitrified 44.5% (N=393) • Conventional 45.1% (N=335)

116. a,b Values within columns without common superscripts differ (P<0.05). Expansion and hatching rates of vitrified embryos Equilibration Time (min) Blastocyst % expansion % hatching 1 60 a 29 a 2 66 a 41 ab 3 82 b 60 b Non-vitrified control 84 b 45 b

117. 1- vs 2-Step Vitrification Treatment % Survival (of controls) 1-step 85  10 2-step 98  10 Thaw temp 24  C 90  10 37  C 93  10

118. Expansion and hatching rates of vitrified in vivo GRADE 1 and 2 embryos Stage (n) % % Re-expansion Hatching Morula (25) 100 96 Blastocysts (20) 95 95

119. Comparison of Macromolecules 2.3 66% + 2.0 91% + + 2.2 61% + Quality Survival SH PVA BSA

121. Straw Configuration DHCDM V2HCDM Cotton plug Plunged end

122. Thawing  In air 10 s  Water bath 37 °C 20 s  Shake the straw  Back in bath  Dry  Embryo transfer

124. Thank you Muchas Gracias