128 Pregnancy rates using in vitro ovine embryos frozen in ethylene glycol and glycerol
A. Velázquez-Roque A , H. Álvarez-Gallardo B , F. Villaseñor-González C , M. Kjelland D E and S. Romo FA Private practice, H&A Biotecnologías en Reproducción Animal, Tepatitlán, Jalisco, México
B Centro Nacional de Recursos Genéticos, INIFAP, Tepatitlán, Jalisco, México
C Campo Experimental Centro Altos de Jalisco, INIFAP, Tepatitlán, Jalisco, México
D Conservation, Genetics & Biotech, LLC, Valley City, North Dakota, USA
E Mayville State University, Mayville, North Dakota, USA
F Facultad de Estudios Superiores Cuautitlán, UNAM, Cuautitlán, México, México
Reproduction, Fertility and Development 35(2) 191-192 https://doi.org/10.1071/RDv35n2Ab128
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
In vitro embryo production (IVP) allows the production of progeny from non-fertile females, pregnant, lactating, and even dead females. The transfer of cryopreserved embryos is very important for commercialisation and for disseminating valuable genetics. A more efficient IVP cryopreservation protocol is still being sought for ovine embryos. The objective of this research was to compare the pregnancy rates of IVP ovine embryos frozen in ethylene glycol (EG) and glycerol (GLY) after embryo transfer (ET). 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 (IVF-Bioscience™) using ovaries collected from a slaughterhouse (León, México). For the IVM, the cumulus-oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24 h at 38.5°C in 5% CO2 in air, and 100% humidity. The IVF process was conducted with semen from the same ram at a concentration of 2 × 106 sperm/mL, for 18 h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. Cleavage rate was 55.9% ± 2.5, and the percentage of blastocysts on Day 7 was 37.3% ± 1.1. The expanded blastocysts (BX) on Day 7 were divided equally (n = 100) in two groups: (a) Frozen EG, and (b) Frozen GLY. BX 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 with sucrose Vigro™) and GLY (Glycerol Freeze plus with sucrose Vigro™), starting at −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. For surgical ET, 100 ewes (commercial cross-bred) with a body score condition of 2.5 (in a scale from 1–5) were used as recipients and synchronised with a CIDR-G (0.3 g) on Day 0; on Day 12, CIDR-G was removed and a 400 UI dose of equine chorionic gonadotrophin was applied; on Day 19, ET was conducted (only ewes in oestrus were used as recipients). Pregnancy was detected using transrectal ultrasonography at 35 days of gestation, and pregnancy rates were evaluated using the chi-squared procedure on the Jamovi software (version 1.2; The Jamovi project). The results were 54% and 34% for frozen EG, and frozen Gly, respectively, being significantly different (P = 0.044) (Table 1). In conclusion, under the conditions of this research, the IVP ovine embryos frozen in EG produced higher pregnancy rates after ET.
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