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CONGELACION DE CONGELACION DE EMBRIONESEMBRIONES
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Ventajas de la Congelacion de Embriones
• No es necesario transferir los embriones inmediatamente después de colectados.
• No es necesario tener muchas receptoras sincronizadas.
• Permite el transporte de genética a bajo costo.
• Facilita la introducción de una nueva raza y la adaptación de los futuros terneros al nuevo medio ambiente.
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•Disminuye el riesgo de introducir enfermedades exóticas.
•Los embriones pueden ser almacenados mientras se testean los padres por enfermedades o por producción.
•Almacenamiento de genética de razas o especies raras o en extinción.
•Permite mantener la diversidad genética a través de un banco de embriones.
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- 5 to - 7 °C - 30 to - 35 °C
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Constitution of Freezing MediumConstitution of Freezing Medium
1. PBS + antibiotic-antimycotic1. PBS + antibiotic-antimycotic2. Biological proteins – 0.4-20%2. Biological proteins – 0.4-20%3. Cryoprotectants - ~10%3. Cryoprotectants - ~10%
*Glycerol*Glycerol*Ethylene Glycol*Ethylene Glycol
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CryoprotectantsCryoprotectants
Permeating e.g. Glycerol or Permeating e.g. Glycerol or ethylene glycolethylene glycol
Nonpermeating e.g. SucroseNonpermeating e.g. Sucrose
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Protocolo de CongelaciónProtocolo de CongelaciónProtocolo de CongelaciónProtocolo de Congelación
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ColectarColectar
Lavar (10 veces)Lavar (10 veces)
Clasificar los embriones.Clasificar los embriones.
Protocolo de CongelaciónProtocolo de CongelaciónProtocolo de CongelaciónProtocolo de Congelación
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Lavado de embriones• 5 lavados con PBS (1/100 volumen de los
embriones y no mas de 10 embriones)
• 2 lavados con tripsina al 0,2% por 30-60 segundos
• 5 lavados con PBS
• Los Embriones deben Tener la ZP intacta, sin material adherido y ser observados a 50X de magnificación
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Lavado de embriones
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Enfermedades que no se Transmiten a través de la Transferencia de Embriones
Categoría 1• Leucosis Enzootica Bovina• Fiebre Aftosa (Bovino)• Lengua Azul (Bovino)• Brucela Abortus (Bovino)• Encefalitis Espongiforme Bovina (BSE)• Scrapie (Ovino)• IBR (Bovino) (con tripsina)• Pseudorrabia (Porcino) (con tripsina)
IETS, 2004
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Categoría 2• Peste Porcina Clasica• Lengua Azul (ovino)
Categoria 3• DVB• Peste Bovina• Haemophilus Sommus• Virus de la Inmunodeficiencia
Bovina• Campilobacter Fetus (ovino)• Fiebre Aftosa (porcino, ovino y
caprino)• Enfermedad Vesicular Porcina• Adenomatosis Pulmonar (ovinos)
Categoría 4• Micobacterium Bovis• Micobacterium Paratuberculosis• Neospora Caninum• Anaplasmosis Bovina• Herpesvirus Bovino-4• Ureaplasma Micoplasma• Clamidia Psitaci• Virus Parainfluenza 3• Estomatitis vesicular (bovino)• Enfermedad de Akabane (bovino)• E. Coli 09:k99 (bovino)• Lepto Borgpeterseni s, hardjobovis• Enterovirus (bovino)• Maedi-visna (ovino)• Scrapie (Caprino)• Lengua Azul (caprino)• Artritis-encefalitis Caprina• Parvovirus porcino• Leptospirosis (porcino)• Brucela Ovis • Peste Porcina Africana• Circovirus Porcino• Sindrome Reproductivo y Respiratorio porcino
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Cycle day: 6 Cycle day: 6.5 Cycle day: 6.5 Cycle day: 6.5 Cycle day: 6.5Stage Code: 3 Stage Code: 3 Stage Code: 3 Stage Code: 3 Stage Code: 4Quality Code:1 Quality Code:1 Quality Code:2 Quality Code:2 Quality Code:1
Cycle day: 7 Cycle day: 7 Cycle day: 7 Cycle day: 7 Cycle day: 7Stage Code: 4 Stage Code: 4 Stage Code: 4 Stage Code: 4 Stage Code: 4Quality Code:1 Quality Code:1 Quality Code:1 Quality Code:1 Quality Code:1
Cycle day: 7 Cycle day: 7 Cycle day: 7 Cycle day: 7 Cycle day: 7Stage Code: 4 Stage Code: 4 Stage Code: 4 Stage Code: 4 Stage Code: 4Quality Code:2 Quality Code:2 Quality Code:2 Quality Code:2 Quality Code:2
BOVINE EMBRYOS: EXAMPLES OF DEVELOPMENTAL STAGE AND QUALITYPage 1
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BOVINE EMBRYOS: EXAMPLES OF DEVELOPMENTAL STAGE AND QUALITYPage 2
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BOVINE EMBRYOS: EXAMPLES OF DEVELOPMENTAL STAGE AND QUALITYPage 3
Cycle day: 7 Cycle day: 7 Cycle day: 7.5 Cycle day: 7.5 Cycle day: 7.5Stage Code: 5 Stage Code: 6 Stage Code: 6 Stage Code: 6 Stage Code: 6Quality Code:3 Quality Code:1 Quality Code:1 Quality Code:1 Quality Code:2
Cycle day: 7.5 Cycle day: 7.5 Cycle day: 7.5 Cycle day: 7.5 Cycle day: 7.5Stage Code: 7 Stage Code: 7 Stage Code: 7 Stage Code: 7 Stage Code: 7Quality Code:1 Quality Code:1 Quality Code:1 Quality Code:1 Quality Code:2
Cycle day: 8.0 Cycle day: 8.0 Cycle day: 7.0 Cycle day: 7.0 Cycle day: 7.0Stage Code: 8 Stage Code: 8 Stage Code: 4 Stage Code: 4 Stage Code: 4Quality Code:1 Quality Code:1 Quality Code:2 Quality Code:1 Quality Code:1
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Calidad y Estadío de los Embriones Respuesta Posible a la Congelación
Calidad y Estadío de los Embriones Respuesta Posible a la Congelación
Estadío Descripción Grado Respuesta posible3 Mórula temprana 1
2 y 3variablemala
4 Mórula compacta 123
muy buenabuena - regularvariable - mala
5 Blastocisto temprano 123
muy buenabuena - regularvariable - mala
6 Blastocisto 123
muy buenabuena - regularmala
7 BlastocistoExpandido
12
muy buena pero variablebuena pero variable
Stroud y Leibo, 1995
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Estado embrión P/total Valor P %Mórula 259/468ab
0,004
55,40
Blastocisto temprano
275/463a 59,39
Blastocisto 179/344bc 52,03
Blastocisto expandido
23/58c 39,66
Total 1333
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Calidad Embrión
P/total Valor P %
1 693/1235a
0,01
56,11
2 32/65ab 49,23
3 11/33b 33,33
Total 1333
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Cryoprotector
EmbryoCotton plug Plug/label
E511 SM010769 SRN 5G DT 1
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Cryoprotector
EmbryoCotton plug Plug/label
E511 SM010769 SRN 5G DT 1
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Protocolo de CongelaciónProtocolo de CongelaciónProtocolo de CongelaciónProtocolo de Congelación
1. Clasificar los embriones (Grado 1).1. Clasificar los embriones (Grado 1).
2. Colocarlos en Crioprotector 1.5 M y 2. Colocarlos en Crioprotector 1.5 M y cargar la pajuela. cargar la pajuela.
Tiempo Total: 10 minutos.Tiempo Total: 10 minutos.
3. Colocar las pajuelas en la congeladora 3. Colocar las pajuelas en la congeladora
(-6,5 ºC) y dejar 1 a 2 minutos.(-6,5 ºC) y dejar 1 a 2 minutos.
4. Realizar el “seeding”. Dejar 10 minutos4. Realizar el “seeding”. Dejar 10 minutos
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SeedingSeeding
Induction of ice crystals in the sample (break Induction of ice crystals in the sample (break the metastable state of solution)*the metastable state of solution)*
Begins cell dehydration processBegins cell dehydration process
*Critical step*Critical step
Crioprotector
Embryo
E511 SM010769 SRN 5G DT 1
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Protocolo de CongelaciónProtocolo de CongelaciónProtocolo de CongelaciónProtocolo de Congelación
1. Clasificar los embriones (Grado 1).1. Clasificar los embriones (Grado 1).
2. Colocarlos en Crioprotector 1.5 M y cargar la 2. Colocarlos en Crioprotector 1.5 M y cargar la pajuela. pajuela.
Tiempo Total: 10 minutos.Tiempo Total: 10 minutos.
3. Colocar las pajuelas en la congeladora 3. Colocar las pajuelas en la congeladora
(-6,5 ºC) y dejar 1 a 2 minutos.(-6,5 ºC) y dejar 1 a 2 minutos.
4. Realizar el “seeding”. Dejar 10 minutos4. Realizar el “seeding”. Dejar 10 minutos
5. Congelar a 0,6 ºC por minuto hasta - 35 ºC.5. Congelar a 0,6 ºC por minuto hasta - 35 ºC.
6. Sumergirlas en N6. Sumergirlas en N22..
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DESCONGELACIONDESCONGELACION
10 segundos en arie10 segundos en arie30 segundos en agua a 25-30 30 segundos en agua a 25-30 ooCC
Vaciar la Pajuela en una placa de PetriVaciar la Pajuela en una placa de Petri
Remover el GlicerolRemover el Glicerol
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Embryo Thawing & Broken Zonae
• Mouse Bovine• n % n %
• 22oC Water 223 (10.3%) 207 (21.7%)
• 22oC Air 220 (0.01%) 202 (0.02%)
•
• Palasz et al. (1989)
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Transferencia Directa Transferencia Directa de Embriones de Embriones
BovinosBovinos
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Cell Volume Changes with Addition and Cell Volume Changes with Addition and Removal of Removal of GlycerolGlycerol vs vs Ethylene GlycolEthylene Glycol
Diluent Diluent AddedAdded
InfluxInfluxEffluxEfflux
11
Rel
ativ
e C
ell
Rel
ativ
e C
ell
Vo
lum
eV
olu
me
Time in Permeating AdditiveTime in Permeating Additive
00
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1997 Census of Cattle Embryos Frozen and Transferred in Canada and the
USA
Glycerol Ethylene Glycol
Country
No. Frozen
% Pregnant
No. Frozen
% Pregnant % of Total
Canada 1,609 58.0 11,376 59.2 87.6
USA 12,411 56.9 15,433 58.5 55.4
(Leibo and Mapletoft, 1998)
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100
90
80
70
60
50
40
Pre
gnancy
(%
)
Seeding Temperatures of Direct Seeding Temperatures of Direct Transfer EmbryosTransfer Embryos
Seeding Temperature (oC)
-5-4 -6 -7 -8
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100
90
80
70
60
50
40
Pre
gnancy
(%
)
Cooling Rate of Direct Transfer Cooling Rate of Direct Transfer EmbryosEmbryos
Cooling Rate (oC/min) from -7oC to -33oC
0.4 0.5 0.6
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Protocolo de CongelaciónProtocolo de CongelaciónProtocolo de CongelaciónProtocolo de Congelación
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Protocolo de CongelaciónProtocolo de Congelación
• 1. Clasificar los embriones (Grado 1).• 2. Colocarlos en EG 1.5 M y cargar la pajuela. • Tiempo Total: 5 minutos.• 3. Colocar las pajuelas en la congeladora (-6,5 ºC) y
dejar 1 a 2 minutos.• 4. Realizar el “seeding”. Dejar 10 minutos• 5. Congelar a 0,5 ºC por minuto hasta - 35 ºC.
• 6. Sumergirlas en N2.
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Direct Transfer StrawDirect Transfer Straw
Ethylene glycol
EmbryoCotton plug Plug/label
E511 SM010769 SRN 5G DT 1
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Pajuela de Transferencia Directa
Pajuela de Transferencia Directa
Etilenglicol 1,5 MEtilenglicol 1,5 M
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Protocolo de CongelaciónProtocolo de Congelación
• 1. Clasificar los embriones (Grado 1).• 2. Colocarlos en EG 1.5 M y cargar la pajuela. • Tiempo Total: 5 minutos.• 3. Colocar las pajuelas en la congeladora (-6,5 ºC) y
dejar 1 a 2 minutos.• 4. Realizar el “seeding”.• 5. Congelar a 0,5 ºC por minuto hasta - 35 ºC.
• 6. Sumergirlas en N2.
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Effect of Time Between Flushing and Freezing on Pregnancy Rate (Embryos frozen at Em Tran & thawed at Holland
Genetics)Time (min) No. Transfers % Pregnant
0 - 30 85 54.1
31 - 60 418 56.0
61 - 90 722 56.0
91 -120 507 56.2
121 - 150 264 53.0
151 - 180 106 55.7
(Hasler, 2001)
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Protocolo de DescongelaciónProtocolo de Descongelación
• 1. Sacar la pajuela.
• 2. Colocarla en Baño María a 30 ºC por 30
segundos.
• 3. Transferir dentro de los 15 minutos de
descongelado.
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100
90
80
70
60
50
40
Pre
gnancy
(%
)
Warming in Air of Direct Transfer EmbryosWarming in Air of Direct Transfer Embryos
Warming Time (sec) in Air
42 6 8 10
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Rutina de Transferencia Directa de Embriones
Rutina de Transferencia Directa de Embriones
• Trabajar al lado de la manga• 1. Anotar número de receptora y embrión.• 2. Examinar ovarios (palpación o US) y
vaciar el recto. • 3. Anestesia epidural, limpiar zona perineal.• 4. Descongelar el embrión y cargarlo en la
vaina de transferencia.• 5. Transferir en el cuerno ipsilateral al CL lo
más rápido posible.• 6. Chequear planilla y largar.
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Tiempo TETF P/total Valor P %≤180 segundos 251/385
0,42
55,84
>180≤360 segundos
372/655 56,79
>360 segundos 42/82 51,22
Total 1122
Peres Coelho et al., 2007
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Embriones Brahman vs Bos Taurus (Looney et al., 2004)
Biotipo Glicerol Etilenglicol Total
N Pre. % N Pre. % %
Británicas 80 49 61 375 240 64 63.5
Continentales
105 61 58 131 80 61 59.7
Sintéticas con Brahman
103 67 65 231 124 54 57.2
Brahman 67 20 30 203 98 48 43.7
Totales 355 197 55.5 940 542 57.7
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Porcentajes de preñez con embriones congelados o refrigerados por 24-30 h y transferidos en receptoras
cruzas Bos Indicus.
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)
Tribulo et al., 2001
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54.2057.50
52.70 52.40
0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00
100.00
Preñadas
Por
cent
aje
30 days60 days
Refrigerated
n=131
%
Frozen-Thawed DT
n=252
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VitrificationVitrificationVitrumVitrum = Latin for glass= Latin for glassFormation of glassy solid without Formation of glassy solid without
ice crystalsice crystalsVery rapid freezingVery rapid freezingHigh molar level (5 to 7M) of High molar level (5 to 7M) of cryoprotectantscryoprotectantsRequires no freezer (Dewar of Requires no freezer (Dewar of
nitrogen)nitrogen)Can be used with in-straw dilutionCan be used with in-straw dilution
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Advantages of Advantages of Vitrification to the Vitrification to the
PractitionerPractitionerNo freezer requiredNo freezer required
Faster with small no. of embryosFaster with small no. of embryos - -
Shortens day with small no. of embryos or at the Shortens day with small no. of embryos or at the
end of the day with “left over” embryosend of the day with “left over” embryos
Higher survival of IVF-derived embryos Higher survival of IVF-derived embryos
Increased survival of lower quality Increased survival of lower quality
embryosembryos
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Procedures Basal embryo holding
medium
Sucrose or galactose
Need four media• Embryo holding medium• “Low” concentration EG• High concentration EG• Dilution medium
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Addition of Addition of CryoprotectantCryoprotectant
3 minutes in 1st EG solution3 minutes in 1st EG solution
45 seconds in 245 seconds in 2ndnd EG solution EG solution• Draw embryo into straw, seal and Draw embryo into straw, seal and
freezefreeze
• Embryos can be loaded and frozen Embryos can be loaded and frozen approximately 1 per minuteapproximately 1 per minute
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Straw ConfigurationStraw Configuration
DHCDM V2HCDM
Cotton plugPlunged end
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ThawingThawing
In air 10 s
Water bath 37°C 20 s
Shake the straw
Back in bath
Dry
Embryo transfer
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