45 Evaluation of two cryoprotectants for the cryopreservation of ovine embryos produced in vitro
G. Márquez-Márquez A , H. Álvarez-Gallardo B , A. Velázquez-Roque C , M. Kjelland D E , F. Villaseñor-González F and S. Romo GA Private practice, Granos y Servicios Integrales SA de CV, San Juan de los Lagos, Jalisco, México
B Centro Nacional de Recursos Genéticos, INIFAP, Tepatitlán, Jalisco, México
C Private practice, H&A Biotecnologías en Reproducción Animal, Tepatitlán, Jalisco, México
D Conservation, Genetics & Biotech, LLC, Valley City, North Dakota, USA
E Mayville State University, Mayville, North Dakota, USA
F Campo Experimental Centro Altos de Jalisco, INIFAP, Tepatitlán, Jalisco, México
G Facultad de Estudios Superiores Cuautitlán, UNAM, Cuautitlán, México, México
Reproduction, Fertility and Development 35(2) 148-148 https://doi.org/10.1071/RDv35n2Ab45
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
The transfer of cryopreserved embryos has great potential to disseminate valuable genetics in sheep; however, a suitable protocol for cryopreservation of in vitro-produced ovine embryos (IVP) is still being improved. A cryopreservation protocol would facilitate the commercialisation of IVP ovine embryos. The objective of this research was to evaluate slow freezing cryotolerance of IVP embryos using ethylene glycol (EG) and glycerol (GLY) as cryoprotectants. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous in vitro culture system based on IVF-Bioscience™ media, using ovaries collected in a slaughterhouse. On Day 7 of in vitro culture, expanded blastocyst stage embryos (n = 218; 109 for EG, 109 for GLY) quality 1 and 2 (according to the fourth edition of the International Embryo Transfer Society Manual) were subjected to a controlled-rate freezing curve after equilibration for 8 to 10 min in freezing medium with EG (Ethylene Glycol Freeze Plus ViGRO™ [Vetoquinol]) and GLY (Glycerol Freeze plus ViGRO™ [Vetoquinol]), starting in −6°C (seeding) and decreasing 0.5°C min−1 and 0.3°C min−1 for EG and GLY respectively, and ending at −32°C and then plunging directly into liquid nitrogen. The embryos frozen in EG were thawed 10 s in air and then 20 s in water at 30°C and cultured for 48 h at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The embryos frozen in GLY were thawed 10 s in air and then 20 s in water at 30°C and were rehydrated (One Step Thaw Plus ViGRO™ [Vetoquinol]) for 15 min. At the end of this period, embryos were cultured for 48 h at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. Re-expansion and hatching rates were evaluated at 24 and 48 h. Statistical analysis was carried out using the chi-squared procedure on the Jamovi software (version 1.2, The Jamovi project). Re-expansion rates at 24 h were 61.5% and 50.5%; at 48 h, 20.2% and 8.2%, respectively for EG and GLY. The percentages of hatching were 60.6% and 38.5% at 24 h and 5.5% and 8.3% at 48 h, respectively for EG and GLY. There were no significant differences between the groups (P > 0.05) for re-expansion at 24 h and for hatching at 48 h, but there were significant differences for total re-expanded (P < 0.001), total hatched (P = 0.004), re-expansion at 48 h (P = 0.012), and hatching at 24 h (P = 0.001) (Table 1). In conclusion, under the conditions of this research, slow freezing with EG had better survival post-thawing for IVP ovine embryos compared to slow freezing with GLY.
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