Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium

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Published on: August 13, 2020

Enrique Gomez a, *, Susana Carrocera a, David Martín a, Juan Jose Perez-Janez b, Javier Prendes b, Jose Manuel Prendes b, Alejandro Vazquez c, Antonio Murillo a, 1, Isabel Gimeno a, Marta Munoz a
a Centro de Biotecnologia Animal-SERIDA, Camino de Rioseco 1225, Gijon, 33394, Spain, b Cooperativa de Agricultores y Usuarios de Gijon, Carretera Carbonera 2230, Poligono Industrial de Roces 5, Gijon, 33211, Spain, c Asturian Biotechnology, Galeno, 2248, Poligono Industrial de Roces 5, Gijon, 33211, Spain

Abstract

Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application.We analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos, integrating: 1) an ethyleneglycol based system; 2) a culture step without protein; and 3) a synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir ovaries were cultured in groups in BSAcontaining synthetic oviduct fluid with or without 0.1% fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in vitro, and development rates, cell counts and dead cells were analyzed in surviving embryos. V/W improved survival over F/T (live and hatching rates at 2 h, 24 h and 48 h) (P < 0.0001), and FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, % pycnotic and % total dead cells higher (p < 0.05) than their Day-8 counterparts, probably because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7 blastocysts were transferred to heifers in an experimental herd. There were no differences in birth rates with frozen (-FCS [n ¼ 40]: 45%; þFCS [n ¼ 14]: 28%), vitrified (-FCS [n ¼ 47]: 53%; þFCS [n ¼ 11]: 36%) and fresh (-FCS [n ¼ 30]: 47%; þFCS [n ¼ 17]: 53%) embryos. However, frozen embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen (n ¼ 22), vitrified (n ¼ 29) and fresh (n ¼ 22) transfers did not differ in birth weight, gestation length and daily gain weight (P > 0.10). Subsequently, transfer of frozen embryos (n ¼ 29) derived from oocytes collected from live, hormonally stimulated cows in experimental herd, led to pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos produced with BSA were transferred to cows in an on-field trial (frozen [n ¼ 80]; fresh [n ¼ 58]), with no differences in pregnancy rates (days 30e40). Pregnancy and birth rates could not be predicted from in vitro approaches.
The new F/T system yields pregnancy and birth rates comparable to vitrified and fresh embryos without birth overweight. The absence of products of animal origin, defined chemical composition, and direct
transfer entail sanitary, manufacturing and application advantages.

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