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
Presentación guía sencilla en Microsoft Excel.pptx
Clase 6 congelacion
1. Reuben J. Mapletoft* Department of Large Animal Clinical Sciences, WCVM, University of Saskatchewan *Bioniche Animal Health, Belleville, ON, Canada CONGELACION DE EMBRIONES
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
15.
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
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
54. Cell Volume Changes with Addition of a Nonpermeating Cryoprotective Agent 1 Relative Cell Volume Time in Nonpermeating Additive 0 Added Removed Sucrose
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
60.
61.
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
65.
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
68.
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
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)
88.
89.
90.
91.
92.
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
94.
95.
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
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)
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
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)
115.
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
