Comparison of two vitrification processes on survival rates of ovine embryos
- Autores
- Fernandez, Jimena; Bruno Galarraga, María Macarena; Cattaneo, Luciano; Prieto, Claudio; Antuña, Sebastián; Tardivo, Belén; Fontana, Diego Sebastian; Bo, Gabriel; Gibbons, Alejandro Eduardo; Cueto, Marcela Isabel
- Año de publicación
- 2022
- Idioma
- inglés
- Tipo de recurso
- documento de conferencia
- Estado
- versión publicada
- Descripción
- The objective of this study was to evaluate the effect of two vitrification and warming processes on the embryo survival rate of ovine embryos. The experiment was conducted at the Small Ruminants Reproduction Laboratory of the National Institute of Agricultural Technology, Bariloche, Argentina (41°S 70°W), during the breeding season. Twenty-four adult multiparous Merino sheep were superovulated using intravaginal sponges (60 mg of medroxyprogesterone acetate (MAP); Progespon®, Zoetis) for 14 days combined with 280 or 480 IU IM of recombinant equine chorionic gonadotrophin (reCG; Folirec®, Zoovet) at the end of progestagen treatment. The onset of oestrus was detected by means of an adult teaser ram (24?48 h after sponge removal). All embryo donors were inseminated using laparoscopy with frozen/thawed semen (100 × 106 sperm per ewe). The embryos (n = 107) were collected surgically 7 (Day 7) and 8 (Day 8) days after intravaginal sponge withdrawal. A total of 92 embryos suitable for vitrification (morulae = 88; blastocysts = 4) were randomly assigned to two vitrification processes at 25°C: V1 (n = 47), three successive solutions: (i) basic medium (BM) (Flushing medium, Dipla flash plus®, Serendipia) supplemented with 5% fetal bovine serum + 10% glycerol (G) + 0.5 M sucrose for 5 min; (ii) BM + 10% G + 12.5% ethylene glycol (EG) + 0.5 M sucrose for 5 min; (iii) BM + 25% G + 25% EG + 0.5 M sucrose for 30 s; V2 (n = 45), using the same solutions as for V1 but without sucrose. The embryos were then loaded in 1 µL of final solution into a plastic micropipette tip (Paralwall) and plunged into liquid nitrogen. For thawing, the micropipette tips were warmed between the thumb and middle fingers for 10 s, and the vitrified embryos were randomly assigned to two warming processes: D1 (three dilution steps): (i) BM + 12.5% G + 12.5% EG + 0.5 M sucrose; (ii) BM + 0.5 M sucrose; and (iii) BM + 0.25 M sucrose, for 5 min each, to allow the removal of cryoprotectants. Then, the embryos were introduced in two BM solutions for 2.5 min each at 25°C; D2 (two dilution steps): (i) BM + 0.5 M sucrose; and (ii) BM + 0.25 M sucrose, for 5 min each. Finally, the embryos were introduced in two BM solutions for 2.5 min each at 39°C. Therefore, four groups were formed: V1D1 (n = 24), V1D2 (n = 23), V2D1 (n = 24), and V2D2 (n = 21). Embryos were cultured in TCM 199 at 39°C and a 6.5% CO2 atmosphere for 48 h according to the different groups. Embryo survival rate data for vitrification and warming processes were analysed using the chi-squared test. Embryo survival rate was greater for embryos in the V1D1 (37.5%) and V1D2 (43.5%) processes than in the V2D2 (14.3%) process (P < 0.05); V2D1 presented an intermediate value relative to the other groups (25.0%). Also, embryos from Day 8 (n = 37) had a higher embryo survival rate than those from Day 7 (n = 55) (50.4 vs. 20.1%, respectively; P < 0.05). In conclusion, the addition of sucrose to the vitrification solutions improved embryo survival rate, regardless of the warming conditions. Furthermore, the day of embryo development at the time of recovery is essential to obtain good embryo survival rates by vitrification.
Fil: Fernandez, Jimena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina
Fil: Bruno Galarraga, María Macarena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina
Fil: Cattaneo, Luciano. Universidad Nacional del Litoral; Argentina
Fil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Biotecnofe Sociedad Anonima.; Argentina
Fil: Antuña, Sebastián. Biotecnofe Sociedad Anonima.; Argentina
Fil: Tardivo, Belén. Biotecnofe Sociedad Anonima.; Argentina
Fil: Fontana, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Biotecnofe Sociedad Anonima.; Argentina
Fil: Bo, Gabriel. Universidad Nacional de Villa María; Argentina. Instituto de Reproducción Animal Córdoba; Argentina
Fil: Gibbons, Alejandro Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina
Fil: Cueto, Marcela Isabel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina
48th Annual Conference International Embryo Technology Society
Savannah
Georgia
International Embryo Technology Society - Materia
-
VITRIFICATION
EMBRYO SURVIVAL
SUCROSE
SHEEP - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
.jpg)
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/225250
Ver los metadatos del registro completo
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Comparison of two vitrification processes on survival rates of ovine embryosFernandez, JimenaBruno Galarraga, María MacarenaCattaneo, LucianoPrieto, ClaudioAntuña, SebastiánTardivo, BelénFontana, Diego SebastianBo, GabrielGibbons, Alejandro EduardoCueto, Marcela IsabelVITRIFICATIONEMBRYO SURVIVALSUCROSESHEEPhttps://purl.org/becyt/ford/4.4https://purl.org/becyt/ford/4The objective of this study was to evaluate the effect of two vitrification and warming processes on the embryo survival rate of ovine embryos. The experiment was conducted at the Small Ruminants Reproduction Laboratory of the National Institute of Agricultural Technology, Bariloche, Argentina (41°S 70°W), during the breeding season. Twenty-four adult multiparous Merino sheep were superovulated using intravaginal sponges (60 mg of medroxyprogesterone acetate (MAP); Progespon®, Zoetis) for 14 days combined with 280 or 480 IU IM of recombinant equine chorionic gonadotrophin (reCG; Folirec®, Zoovet) at the end of progestagen treatment. The onset of oestrus was detected by means of an adult teaser ram (24?48 h after sponge removal). All embryo donors were inseminated using laparoscopy with frozen/thawed semen (100 × 106 sperm per ewe). The embryos (n = 107) were collected surgically 7 (Day 7) and 8 (Day 8) days after intravaginal sponge withdrawal. A total of 92 embryos suitable for vitrification (morulae = 88; blastocysts = 4) were randomly assigned to two vitrification processes at 25°C: V1 (n = 47), three successive solutions: (i) basic medium (BM) (Flushing medium, Dipla flash plus®, Serendipia) supplemented with 5% fetal bovine serum + 10% glycerol (G) + 0.5 M sucrose for 5 min; (ii) BM + 10% G + 12.5% ethylene glycol (EG) + 0.5 M sucrose for 5 min; (iii) BM + 25% G + 25% EG + 0.5 M sucrose for 30 s; V2 (n = 45), using the same solutions as for V1 but without sucrose. The embryos were then loaded in 1 µL of final solution into a plastic micropipette tip (Paralwall) and plunged into liquid nitrogen. For thawing, the micropipette tips were warmed between the thumb and middle fingers for 10 s, and the vitrified embryos were randomly assigned to two warming processes: D1 (three dilution steps): (i) BM + 12.5% G + 12.5% EG + 0.5 M sucrose; (ii) BM + 0.5 M sucrose; and (iii) BM + 0.25 M sucrose, for 5 min each, to allow the removal of cryoprotectants. Then, the embryos were introduced in two BM solutions for 2.5 min each at 25°C; D2 (two dilution steps): (i) BM + 0.5 M sucrose; and (ii) BM + 0.25 M sucrose, for 5 min each. Finally, the embryos were introduced in two BM solutions for 2.5 min each at 39°C. Therefore, four groups were formed: V1D1 (n = 24), V1D2 (n = 23), V2D1 (n = 24), and V2D2 (n = 21). Embryos were cultured in TCM 199 at 39°C and a 6.5% CO2 atmosphere for 48 h according to the different groups. Embryo survival rate data for vitrification and warming processes were analysed using the chi-squared test. Embryo survival rate was greater for embryos in the V1D1 (37.5%) and V1D2 (43.5%) processes than in the V2D2 (14.3%) process (P < 0.05); V2D1 presented an intermediate value relative to the other groups (25.0%). Also, embryos from Day 8 (n = 37) had a higher embryo survival rate than those from Day 7 (n = 55) (50.4 vs. 20.1%, respectively; P < 0.05). In conclusion, the addition of sucrose to the vitrification solutions improved embryo survival rate, regardless of the warming conditions. Furthermore, the day of embryo development at the time of recovery is essential to obtain good embryo survival rates by vitrification.Fil: Fernandez, Jimena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; ArgentinaFil: Bruno Galarraga, María Macarena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; ArgentinaFil: Cattaneo, Luciano. Universidad Nacional del Litoral; ArgentinaFil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Biotecnofe Sociedad Anonima.; ArgentinaFil: Antuña, Sebastián. Biotecnofe Sociedad Anonima.; ArgentinaFil: Tardivo, Belén. Biotecnofe Sociedad Anonima.; ArgentinaFil: Fontana, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Biotecnofe Sociedad Anonima.; ArgentinaFil: Bo, Gabriel. Universidad Nacional de Villa María; Argentina. Instituto de Reproducción Animal Córdoba; ArgentinaFil: Gibbons, Alejandro Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; ArgentinaFil: Cueto, Marcela Isabel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina48th Annual Conference International Embryo Technology SocietySavannahGeorgiaInternational Embryo Technology SocietyCsiro Publishing2022info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectConferenciaJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/225250Comparison of two vitrification processes on survival rates of ovine embryos; 48th Annual Conference International Embryo Technology Society; Savannah; Georgia; 2022; 249-2501031-3613CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.publish.csiro.au/rd/fulltext/RDv34n2Ab30info:eu-repo/semantics/altIdentifier/doi/10.1071/RDv34n2Ab30Internacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:50:45Zoai:ri.conicet.gov.ar:11336/225250instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:50:45.282CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
| dc.title.none.fl_str_mv |
Comparison of two vitrification processes on survival rates of ovine embryos |
| title |
Comparison of two vitrification processes on survival rates of ovine embryos |
| spellingShingle |
Comparison of two vitrification processes on survival rates of ovine embryos Fernandez, Jimena VITRIFICATION EMBRYO SURVIVAL SUCROSE SHEEP |
| title_short |
Comparison of two vitrification processes on survival rates of ovine embryos |
| title_full |
Comparison of two vitrification processes on survival rates of ovine embryos |
| title_fullStr |
Comparison of two vitrification processes on survival rates of ovine embryos |
| title_full_unstemmed |
Comparison of two vitrification processes on survival rates of ovine embryos |
| title_sort |
Comparison of two vitrification processes on survival rates of ovine embryos |
| dc.creator.none.fl_str_mv |
Fernandez, Jimena Bruno Galarraga, María Macarena Cattaneo, Luciano Prieto, Claudio Antuña, Sebastián Tardivo, Belén Fontana, Diego Sebastian Bo, Gabriel Gibbons, Alejandro Eduardo Cueto, Marcela Isabel |
| author |
Fernandez, Jimena |
| author_facet |
Fernandez, Jimena Bruno Galarraga, María Macarena Cattaneo, Luciano Prieto, Claudio Antuña, Sebastián Tardivo, Belén Fontana, Diego Sebastian Bo, Gabriel Gibbons, Alejandro Eduardo Cueto, Marcela Isabel |
| author_role |
author |
| author2 |
Bruno Galarraga, María Macarena Cattaneo, Luciano Prieto, Claudio Antuña, Sebastián Tardivo, Belén Fontana, Diego Sebastian Bo, Gabriel Gibbons, Alejandro Eduardo Cueto, Marcela Isabel |
| author2_role |
author author author author author author author author author |
| dc.subject.none.fl_str_mv |
VITRIFICATION EMBRYO SURVIVAL SUCROSE SHEEP |
| topic |
VITRIFICATION EMBRYO SURVIVAL SUCROSE SHEEP |
| purl_subject.fl_str_mv |
https://purl.org/becyt/ford/4.4 https://purl.org/becyt/ford/4 |
| dc.description.none.fl_txt_mv |
The objective of this study was to evaluate the effect of two vitrification and warming processes on the embryo survival rate of ovine embryos. The experiment was conducted at the Small Ruminants Reproduction Laboratory of the National Institute of Agricultural Technology, Bariloche, Argentina (41°S 70°W), during the breeding season. Twenty-four adult multiparous Merino sheep were superovulated using intravaginal sponges (60 mg of medroxyprogesterone acetate (MAP); Progespon®, Zoetis) for 14 days combined with 280 or 480 IU IM of recombinant equine chorionic gonadotrophin (reCG; Folirec®, Zoovet) at the end of progestagen treatment. The onset of oestrus was detected by means of an adult teaser ram (24?48 h after sponge removal). All embryo donors were inseminated using laparoscopy with frozen/thawed semen (100 × 106 sperm per ewe). The embryos (n = 107) were collected surgically 7 (Day 7) and 8 (Day 8) days after intravaginal sponge withdrawal. A total of 92 embryos suitable for vitrification (morulae = 88; blastocysts = 4) were randomly assigned to two vitrification processes at 25°C: V1 (n = 47), three successive solutions: (i) basic medium (BM) (Flushing medium, Dipla flash plus®, Serendipia) supplemented with 5% fetal bovine serum + 10% glycerol (G) + 0.5 M sucrose for 5 min; (ii) BM + 10% G + 12.5% ethylene glycol (EG) + 0.5 M sucrose for 5 min; (iii) BM + 25% G + 25% EG + 0.5 M sucrose for 30 s; V2 (n = 45), using the same solutions as for V1 but without sucrose. The embryos were then loaded in 1 µL of final solution into a plastic micropipette tip (Paralwall) and plunged into liquid nitrogen. For thawing, the micropipette tips were warmed between the thumb and middle fingers for 10 s, and the vitrified embryos were randomly assigned to two warming processes: D1 (three dilution steps): (i) BM + 12.5% G + 12.5% EG + 0.5 M sucrose; (ii) BM + 0.5 M sucrose; and (iii) BM + 0.25 M sucrose, for 5 min each, to allow the removal of cryoprotectants. Then, the embryos were introduced in two BM solutions for 2.5 min each at 25°C; D2 (two dilution steps): (i) BM + 0.5 M sucrose; and (ii) BM + 0.25 M sucrose, for 5 min each. Finally, the embryos were introduced in two BM solutions for 2.5 min each at 39°C. Therefore, four groups were formed: V1D1 (n = 24), V1D2 (n = 23), V2D1 (n = 24), and V2D2 (n = 21). Embryos were cultured in TCM 199 at 39°C and a 6.5% CO2 atmosphere for 48 h according to the different groups. Embryo survival rate data for vitrification and warming processes were analysed using the chi-squared test. Embryo survival rate was greater for embryos in the V1D1 (37.5%) and V1D2 (43.5%) processes than in the V2D2 (14.3%) process (P < 0.05); V2D1 presented an intermediate value relative to the other groups (25.0%). Also, embryos from Day 8 (n = 37) had a higher embryo survival rate than those from Day 7 (n = 55) (50.4 vs. 20.1%, respectively; P < 0.05). In conclusion, the addition of sucrose to the vitrification solutions improved embryo survival rate, regardless of the warming conditions. Furthermore, the day of embryo development at the time of recovery is essential to obtain good embryo survival rates by vitrification. Fil: Fernandez, Jimena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina Fil: Bruno Galarraga, María Macarena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina Fil: Cattaneo, Luciano. Universidad Nacional del Litoral; Argentina Fil: Prieto, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Biotecnofe Sociedad Anonima.; Argentina Fil: Antuña, Sebastián. Biotecnofe Sociedad Anonima.; Argentina Fil: Tardivo, Belén. Biotecnofe Sociedad Anonima.; Argentina Fil: Fontana, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Biotecnofe Sociedad Anonima.; Argentina Fil: Bo, Gabriel. Universidad Nacional de Villa María; Argentina. Instituto de Reproducción Animal Córdoba; Argentina Fil: Gibbons, Alejandro Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina Fil: Cueto, Marcela Isabel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche. Instituto de Investigaciones Forestales y Agropecuarias Bariloche. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina 48th Annual Conference International Embryo Technology Society Savannah Georgia International Embryo Technology Society |
| description |
The objective of this study was to evaluate the effect of two vitrification and warming processes on the embryo survival rate of ovine embryos. The experiment was conducted at the Small Ruminants Reproduction Laboratory of the National Institute of Agricultural Technology, Bariloche, Argentina (41°S 70°W), during the breeding season. Twenty-four adult multiparous Merino sheep were superovulated using intravaginal sponges (60 mg of medroxyprogesterone acetate (MAP); Progespon®, Zoetis) for 14 days combined with 280 or 480 IU IM of recombinant equine chorionic gonadotrophin (reCG; Folirec®, Zoovet) at the end of progestagen treatment. The onset of oestrus was detected by means of an adult teaser ram (24?48 h after sponge removal). All embryo donors were inseminated using laparoscopy with frozen/thawed semen (100 × 106 sperm per ewe). The embryos (n = 107) were collected surgically 7 (Day 7) and 8 (Day 8) days after intravaginal sponge withdrawal. A total of 92 embryos suitable for vitrification (morulae = 88; blastocysts = 4) were randomly assigned to two vitrification processes at 25°C: V1 (n = 47), three successive solutions: (i) basic medium (BM) (Flushing medium, Dipla flash plus®, Serendipia) supplemented with 5% fetal bovine serum + 10% glycerol (G) + 0.5 M sucrose for 5 min; (ii) BM + 10% G + 12.5% ethylene glycol (EG) + 0.5 M sucrose for 5 min; (iii) BM + 25% G + 25% EG + 0.5 M sucrose for 30 s; V2 (n = 45), using the same solutions as for V1 but without sucrose. The embryos were then loaded in 1 µL of final solution into a plastic micropipette tip (Paralwall) and plunged into liquid nitrogen. For thawing, the micropipette tips were warmed between the thumb and middle fingers for 10 s, and the vitrified embryos were randomly assigned to two warming processes: D1 (three dilution steps): (i) BM + 12.5% G + 12.5% EG + 0.5 M sucrose; (ii) BM + 0.5 M sucrose; and (iii) BM + 0.25 M sucrose, for 5 min each, to allow the removal of cryoprotectants. Then, the embryos were introduced in two BM solutions for 2.5 min each at 25°C; D2 (two dilution steps): (i) BM + 0.5 M sucrose; and (ii) BM + 0.25 M sucrose, for 5 min each. Finally, the embryos were introduced in two BM solutions for 2.5 min each at 39°C. Therefore, four groups were formed: V1D1 (n = 24), V1D2 (n = 23), V2D1 (n = 24), and V2D2 (n = 21). Embryos were cultured in TCM 199 at 39°C and a 6.5% CO2 atmosphere for 48 h according to the different groups. Embryo survival rate data for vitrification and warming processes were analysed using the chi-squared test. Embryo survival rate was greater for embryos in the V1D1 (37.5%) and V1D2 (43.5%) processes than in the V2D2 (14.3%) process (P < 0.05); V2D1 presented an intermediate value relative to the other groups (25.0%). Also, embryos from Day 8 (n = 37) had a higher embryo survival rate than those from Day 7 (n = 55) (50.4 vs. 20.1%, respectively; P < 0.05). In conclusion, the addition of sucrose to the vitrification solutions improved embryo survival rate, regardless of the warming conditions. Furthermore, the day of embryo development at the time of recovery is essential to obtain good embryo survival rates by vitrification. |
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2022 |
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Comparison of two vitrification processes on survival rates of ovine embryos; 48th Annual Conference International Embryo Technology Society; Savannah; Georgia; 2022; 249-250 1031-3613 CONICET Digital CONICET |
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