138 DEVELOPMENTAL COMPETENCE OF BIOPSIED AND SPLIT BOVINE EMBRYOS
M. Reichenbach A , S. Jung B , R. Fries B , E. Wolf C , C. Gschoederer A , J. Scherzer A , T. Grupp A and H.-D. Reichenbach DA Bayern-Genetik GmbH, Grub, Bavaria, Germany;
B Chair for Animal Breeding, Technische Universität München, Freising, Bavaria, Germany;
C Chair for Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Bavaria, Germany;
D Bavarian State Research Center for Agriculture, Institute of Animal Breeding, Grub, Bavaria, Germany
Reproduction, Fertility and Development 27(1) 161-161 https://doi.org/10.1071/RDv27n1Ab138
Published: 4 December 2014
Abstract
The aim of the present study was to develop a reliable method to simultaneously split and biopsy valuable bovine embryos for a complete genomic evaluation (gender, polledness, and hereditary abnormalities) and to estimate the breeding value of progeny for traits of economic importance immediately after embryo recovery. A total of 208 good quality embryos collected from superovulated German Simmental animals were biopsied immediately after recovery using an inverse microscope (Zeiss, Germany) at 50× magnification with a single-use steel blade mounted on a holder (Bausch & Lomb, Germany) attached to a micromanipulator (Eppendorf, Germany). Biopsy was performed either by splitting the embryo and cutting of one-third of a half [G1: morulae (M), n = 50; early blastocysts (EB), n = 24; blastocysts (B), n = 16], by just splitting in equal halves (G2: M, n = 16; B, n = 2), or by cutting of just a small biopsy of the embryo (G3: M, n = 53) or of the trophoblast (G3: EB, n = 19; B, n = 28). Biopsied cells were immediately used for DNA amplification. Biopsied embryos (E) and demi-embryos (DE) were in vitro cultured in SOF, under mineral oil, at 39°C and 5% CO2, 5% O2, 90% N2 for 24 h, after which survival was recorded. Survival rate of G1 (survival of at least 1 DE: M, 98.0%; EB, 100.0%; B, 93.8%), G2 (survival of DE: M, 75.0%; B, 100.0%), and G3 (embryo survival: M, 96.3%; EB, 100.0%; B, 96.4%) were similar, but in relation to the number of original embryo the highest ratio of DE was obtained in G1 (1.67) v. G2 (0.88) and G3 (0.97; G1:G2/G3; P < 0.01). Within G1, the highest ration to the original number of embryos was by using M (1.78), followed by EB (1.75) and B (1.19; M/EB:B; P < 0.05). To verify the viability of biopsied embryos some DE from G1 (1, the nonbiopsied DE, n = 7, or 2, the biopsied and the nonbiopsied DE per recipient, n = 21), G2 (1 DE per recipient, n = 13), and G3 (1 E per recipient, n = 8) were transferred after 24 h of culture. Overall pregnancy rate (Day 42) of G1, G2, and G3 was 64.3, 23.1, and 50.0%, respectively (G1 : G2; P < 0.05). In G1, pregnancy rates (Day 42) of biopsied embryos differed significantly if either 1 or 2 DE were transferred per recipient (28.6 v. 76.2%, respectively; P < 0.05). A twin pregnancy rate of 38.9% was observed by ultrasonography in recipients when 2 DE were transferred. The results suggest that high survival rates can be obtained with the G1 technique, and splitting during biopsy can increase productivity in programs aimed to evaluate the genomic constitution of early stage embryos.
Funded by the Bayerische Forschungsstiftung (AZ-1031-12).
