126 TIME OF FIRST CLEAVAGE AND DEVELOPMENT TO BLASTOCYST OF BOVINE IVF EMBRYOS MONITORED BY TIME-LAPSE IMAGING AFTER IN VITRO OR IN VIVO MATURATION
A. Beck A , M. Reichenbach B , H. D. Reichenbach C , F. Habermann D , G. J. Arnold E and E. Wolf A BA Molecular Animal Breeding and Biotechnology, Oberschleissheim, Germany;
B Bavarian Research Centre for Biology of Reproduction, Oberschleissheim, Germany;
C Institute for Animal Breeding, Bavarian State Research Center for Agriculture, Grub, Germany;
D Veterinary Anatomy, Histology and Embryology LMU, Munich, Germany;
E LAFUGA, Gene Center, Munich, Germany
Reproduction, Fertility and Development 25(1) 210-210 https://doi.org/10.1071/RDv25n1Ab126
Published: 4 December 2012
Abstract
As part of a study on structural, molecular, and functional deficiencies of both in vitro and in vivo matured oocytes, the outcome of in vitro fertilization and embryo culture was evaluated by noninvasive time-lapse monitoring. Oocytes for IVM were recovered from abattoir-derived ovaries (G1). In vivo matured oocytes were obtained by ovum pick-up from six FSH (Pluset®, Laboratorios Calier, Spain) superstimulated Simmental heifers (Reichenbach et al. 2010 Reprod Domest Anim. 45, 41) shortly before ovulation (G2). Gonadotropin-releasing hormone (GnRH; Receptal®, MSD, Germany) was applied 18 h before ovum pickup (OPU). After IVM (G1) for 23 h or directly after OPU (G2), all oocytes were fertilized and cultured in vitro for 7 days. To continuously monitor multiple individual embryos in parallel, a well-of-wells culture dish system and a transmitted-light microscope equipped with a digital camera placed in the incubator (Primo Vision, Cryo Innovation Inc., Budapest, Hungary) was used. Each embryo was photographed every 5 min over the whole culture period to precisely determine the time and morphological pattern of the first cleavage and to observe the development up to the hatching blastocyst stage. In a first set of experiments, 128 (G1) and 64 (G2) embryos were cultured and monitored under identical conditions. In a statistical analysis (ANOVA, SAS, SAS Institute Inc., Cary, NC, USA) only embryos with a morphologically regular first and second cleavage (G1: n = 86; G2; n = 42; Total n = 128) were included. Fixed effects in the model were time of first cleavage (class 1: ≤26 hours post-insemination (hpi), class 2: >26 to 28 hpi, class 3: >28 to 30 hpi, class 4: >30 to 32 hpi, class 5: >32 hpi) and in vitro versus ex vivo maturation on blastocyst outcome. The total blastocyst rate was lower after in vitro maturation (40/86) than after in vivo maturation (29/42), while the time of first cleavage was not different. The time until the onset of the first cytokinesis ranged in both oocyte groups from 25–37 hpi. Notably, zygotes that cleaved between 26 and 28 hpi (class 2) showed the highest blastocyst rate (35/47), while the rate decreased in a statistically significant way (P < 0.01), when the first cleavage was observed between 30 and 32 hpi (class 4) (4/15). Now we turn to the investigation of the causes of a delay and the aberrations of the first cleavage cycle. An important step is the use of noninvasive microscopic monitoring to select embryos according to their developmental stage and history for 3D fluorescence microscopy, proteome analyses, and functional studies.
This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR1041).
